Suppression of SNARE‐dependent exocytosis in retinal glial cells and its effect on ischemia‐induced neurodegeneration

Nervous tissue is characterized by a tight structural association between glial cells and neurons. It is well known that glial cells support neuronal functions, but their role under pathologic conditions is less well understood. Here, we addressed this question in vivo using an experimental model of retinal ischemia and transgenic mice for glia‐specific inhibition of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE)‐dependent exocytosis. Transgene expression reduced glutamate, but not ATP release from single Müller cells, impaired glial volume regulation under normal conditions and reduced neuronal dysfunction and death in the inner retina during the early stages of ischemia. Our study reveals that the SNARE‐dependent exocytosis in glial cells contributes to neurotoxicity during ischemia in vivo and suggests glial exocytosis as a target for therapeutic approaches.     

Inhibition of Müller cell glutamine synthetase rapidly impairs the retinal response to light

It is widely assumed that neurones have sufficient metabolic reserves to allow them to function independently of glial cells for extended periods. The present study investigates the length of time taken before retinal neurones no longer respond normally to light after the inhibition of glial enzymes that are involved in the synthesis of precursors of neuronal glutamate. The glutamine synthetase inhibitor methionine sulfoximine, when injected intraocularly in Wistar rats, caused a time- and dose-dependent suppression of the scotopic electroretinogram b-wave. At the highest dosage (40 mM) the b-wave was significantly reduced within 2 min of injection. Because the b-wave is an indicator of neurotransmission in the retina, it is deduced that inhibition of glutamine synthetase rapidly blocks glutamatergic neurotransmission. Immunohistochemistry revealed a depletion of neuronal glutamate and an accumulation of glutamate in Müller glial cells, in a time course that matched the b-wave suppression. The b-wave was quickly restored by injection of glutamine (4 mM). The rapid reduction of glutamatergic transmission after methionine sulfoximine administration challenges the view that neurones have sufficient reserves to allow them to function independently for extended periods; instead, it indicates that glia are essential for the moment-to-moment sustenance of neuronal function.     

Peripapillary glial cells in the chick retina: A special glial cell type expressing astrocyte, radial glia, neuron, and oligodendrocyte markers throughout development

Peripapillary glial cells of the chick are a special type of glia, not only because of their position, forming a boundary between the retina on one side and the optic nerve head (ONH) and the pecten on the other, but also because although they have the same orientation and similar shape as the retinal Müller cell (a type of radial glia) and express common markers for these cells and astrocytes, they do not express glutamine synthetase (GS) or carbonic anhydrase C (CA-C), enzymes intensely expressed by Müller cells and astrocytes. In this study, we present further molecular characterization of these cells, using immunohistochemistry techniques. We show that peripapillary glial cells express a novel neuron antigen, 3BA8, that in the adult retina is located only in one neuron type (the amacrine cell) and in the inner plexiform layer (IPL). They also express an antigen specific to myelin and oligodendrocytes, MOSP, and a glial antigen, 3CB2, expressed by radial glia and astrocytes throughout the CNS. The study of the developmental expression of these three antigens in the peripapillary glial cell territory shows different spatiotemporal labeling patterns: 3CB2 and 3BA8 are expressed much earlier (embryonic days E3 and E5, respectively) than MOSP (E12), and during a developmental window (E6-E10) 3BA8 labels the peripapillary glial cells intensely and does not label the ONH or the optic nerve (ON), which are labeled later. The expression of 3CB2 is much more intense in the peripapillary glial cells than in Müller cells from early stages of development up to E16, and the expression of MOSP starts earlier in the peripapillary glial cells than in the Müller cells and is maintained with much higher intensity in the peripapillary glial cells throughout development. These findings show that Müller and peripapillary glial cells follow independent courses of differentiation, which together with the fact that the peripapillary glial cells express molecules typical of neurons, oligodendrocytes, radial glia, and astrocytes are evidence that peripapillary glial cells are a unique type of glia in the CNS.     

Oxidative stress promotes proliferation and dedifferentiation of retina glial cells in vitro

Oxidative damage is involved in triggering neuronal death in several retinal neurodegenerative diseases. The recent finding of stem cells in the retina suggests that both preventing neuronal death and replacing lost neurons might be useful strategies for treating these diseases. We have previously shown that oxidative stress induces apoptosis in cultured retinal neurons. We now investigated the response of Müller cells, proposed as retina stem cells, to this damage. Treatment of glial cell cultures prepared from rat retinas with the oxidant paraquat (PQ) did not induce glial cell apoptosis. Instead, PQ promoted their rapid dedifferentiation and proliferation. PQ decreased expression of a marker of differentiated glial cells, simultaneously increasing the expression of smooth muscle actin, shown to increase with glial dedifferentiation, the levels of cell-cycle markers, and the number of glial cells in the cultures. In addition, glial cells protected neurons in coculture from apoptosis induced by PQ and H(2)O(2). In pure neuronal cultures, PQ induced apoptosis of photoreceptors and amacrine neurons, simultaneously decreasing the percentage of neurons preserving mitochondrial membrane potential; coculturing neurons with glial cells completely prevented PQ-induced apoptosis and preserved mitochondrial potential in both neuronal types. These results demonstrate that oxidative damage activated different responses in Müller glial cells; they rapidly dedifferentiated and enhanced their proliferation, concurrently preventing neuronal apoptosis. Glial cells might not only preserve neuronal survival but also activate their cell cycle in order to provide a pool of new progenitor cells that might eventually be manipulated to preserve retinal functionality.     

Tetanus toxin binding to isolated and cultured rat retinal glial cells

The presence of immunocytochemically detectable membrane receptors for tetanus toxin, supposedly composed of higher gangliosides, is widely accepted as a marker of neuronal cells. We now demonstrate that Müller cells, a unique glial cell type of the vertebrate retina, possess specific tetanus toxin (TT)-binding sites. Single cell suspensions were prepared from adult rat retina by a gentle dissociation method, and the Müller cells, unequivocally identified by their morphology, could be immunocytochemically double-labeled by antisera to vimentin and to TT. The expression of complex gangliosides by identified Müller cells was also demonstrated by immunofluorescence labeling with the monoclonal antibody A2B5. Using the double-immunolabeling method for the identification of Müller cells we show that specific tetanus toxin binding is acquired by these cells during postnatal maturation both in vivo and in vitro. In vivo the percentage of tetanus toxin-positive Müller cells increases from 0% in 4-day-old animals to 10% on postnatal day 8, reaching the adult level of about 95-100% around day 30. In retinal monolayer cultures prepared from newborn rats, the majority (65%) of vimentin-positive non-neuronal cells became TT-positive during a 2-week culture period, indicating that this population of non-neuronal cells represents differentiating Müller cells. Again, comparable results were obtained with A2B5, supporting the conclusion that Müllerian glia expresses surface molecules, which are normally regarded as neuronal markers.     

Neuronal and glial differentiation during lizard (Gallotia galloti) visual system ontogeny

We studied the histogenesis of the lizard visual system (E30 to adulthood) by using a selection of immunohistochemical markers that had proved relevant for other vertebrates. By E30, the Pax6(+) pseudostratified retinal epithelium shows few newborn retinal ganglion cells (RGCs) in the centrodorsal region expressing neuron- and synaptic-specific markers such as betaIII-tubulin (Tuj1), synaptic vesicle protein-2 (SV2), and vesicular glutamate transporter-1 (VGLUT1). Concurrently, pioneer RGC axons run among the Pax2(+) astroglia in the optic nerve and reach the superficial optic tectum. Between E30 and E35, the optic chiasm and optic tract remain acellular, but the latter contains radial processes with subpial endfeet expressing vimentin (Vim). From E35, neuron- and synaptic-specific stainings spread in the retina and optic tectum, whereas retinal Pax6, and Tuj1/SV2 in RGC axons decrease. Müller glia and abundant optic nerve glia express a variety of glia-specific markers until adulthood. Subpopulations of optic nerve glia are also VGLUT1(+) and cluster differentiation-44 (CD44)-positive but cytokeratin-negative, unlike the case in other regeneration-competent species. Specifically, coexpression of CD44/Vim and glutamine synthetase (GS)/VGLUT1 reflects glial specialization, insofar as most CD44(+) glia are GS(-). In the adult optic tract and tectum, radial glia and free astroglia coexist. The latter show different immunocharacterization (Pax2(-)/CD44(-) /Vim(-)) compared with that in the optic nerve. We conclude that upregulation of Tuj1 and SV2 is required for axonal outgrowth and search for appropriate targets, whereas Pax2(+) optic nerve astroglia and Vim(+) radial glia may aid in early axonal guidance. Spontaneous axonal regrowth seems to succeed despite the heterogeneous mammalian-like glial environment in the lizard optic nerve.     

Amyloid beta peptide causes chronic glial cell activation and neuro-degeneration after intravitreal injection

We have previously demonstrated that amyloid beta (Abeta) peptide is acutely toxic to retinal neurones in vivo and that this toxicity is mediated by an indirect mechanism. We have now extended these studies to look at the chronic effect of intravitreal injection of Abeta peptides on retinal ganglion cells (RGC), the projection neurones of the retina and the glial cell response. 5 months after injection of Abeta1-42 or Abeta42-1 there was no significant reduction in RGC densities but there was a significant reduction in the retinal surface area after both peptides. Phosphate-buffered saline (PBS) injection had no effect on retinal size or RGC density. There was a pronounced reduction in the number of large RGCs with a concomitant significant increase in medium and small RGCs. There was no change in cell sizes 5 months after injection with PBS. At 5 months after injection of both peptides, there was marked activation of Muller glial cells and microglia. There was also expression of the major histocompatibility complex (MHC) class II molecule on some of the microglial cells but we saw no evidence of T-cell infiltration into the injected retinas. In order to elucidate potential toxic mechanisms, we have looked at levels of glutamine synthetase and nitric oxide synthase. As early as 2 days after injection we noted that activation of Muller glia was associated with a decrease in glutamine synthetase immuno-reactivity but there was no detectable expression of inducible nitric oxide synthase in any retinal cells. These results suggest that chronic activation of glial cells induced by Abeta peptides may result in chronic atrophy of projection neurones in the rat retina.     

Growth regulator from spinal cord: produced in cultures of glial cells

Spinal cord cultures produced a regulatory factor which inhibited myogenesis. After serial passage (x 3) production of this factor continued as neuronal cells disappeared and large, pale polygonal cells rich in cytoplasmic microfilaments with morphology of astrocyte precursors became predominant. These glial cells responded to dibutyryl cyclic AMP by assuming a star-shaped appearance with multiple, radiating cytoplasmic processes. Cultures active in production of the growth regulator also produced nonneuronal-type enolase and glutamine synthetase. It is suggested that the growth regulator is produced by astrocytic glia in culture.     

Taurine uptake by chick embryo retinal neurons and glial cells in purified culture

The properties and cellular distribution of a high-affinity uptake mechanism for taurine have been investigated using separate populations of purified chick embryo neural retina neurons and glia. Purified neuronal monolayers, cultured in serum-free medium, were incubated in radioactive taurine under different conditions and studied autoradiographically and biochemically. Labeling with radioactive taurine was detected in the perikaryon of most of the neurons present in the cultures. Neuronal uptake occurred by means of a high-affinity mechanism which was completely inhibited at low temperatures or in the absence of sodium ions. The uptake was linear for at least 1 hr and, as is the case in vivo, could be inhibited by gamma-aminobutyric acid (GABA) or beta-alanine. Incubation in ouabain, glutamate, or high K+ concentrations failed to cause any increase in the amount of taurine released by neurons preloaded with the radioactive amino acid. The rather wide-spread distribution of high-affinity taurine uptake was confirmed using separate retinal cultures rich in glial cells. Practically 100% of the glial cells appeared labeled after incubation in 10(-7) M [3H] taurine, and this uptake was also inhibited by low-temperature, Na+-free medium, GABA, or beta-alanine. Several pieces of evidence indicate that high-affinity taurine uptake coexists with uptake mechanisms for other amino acids, such as GABA, glutamate, and aspartate, in retinal neurons as well as glial cells. These in vitro populations offer a promising experimental system for the investigation of the effects of taurine on retinal cells.     

Primary glial cells and brain fibroblasts: Interactions in culture

Primary glial-enriched cultures were prepared from newborn mouse cerebral hemispheres. The cultures were grown in Dulbecco's Modified Eagle Medium in which L-valine was substituted with D-valine; this medium selectively inhibits the growth of fibroblasts. Using glutamine synthetase and glial fibrillary acidic protein as immunocytochemical markers, cultures in D-valine medium were characterized as being over 80% astrocytic. However, these cultures exhibited a suppressed growth rate and lagged behind in their differentiation as assessed biochemically using DNA content and glutamine synthetase activity as markers for growth and differentiation. Growth was restored when D-valine cultures were grown in medium containing conditioned medium derived from brain fibroblast cultures when grown on matrix or killed substrata derived from brain fibroblast cultures. This in vitro approach offers the possibility of purifying factors and developing immunological probes to investigate the possible role of brain fibroblasts in influencing glial cell function.     

Differences in neuronal and glial cell phenotypic expression in neuron-glia cocultures: influence of glia-conditioned media and living glial cell substrata.

Neuron-glia cocultures were prepared using, as a source for glial cells, either C6 glia (2B clone) of early (2B23) or late (2B111) passages or advanced passages of glial cells derived from primary cultures prepared from aged mouse cerebral hemispheres (MACH). Six-day-old chick embryo cerebral hemispheres (E6CH) were the source of neuron-enriched cultures. Glutamine synthetase (GS) activity was used as a marker for astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity was used as a marker for oligodendrocytes. GS activity was markedly enhanced in cocultures of E6CH neurons and 2B23 glioblastic cells, whereas GS activity was reduced in cocultures of E6CH neurons and 2B111 astrocytic glia. In contrast, CNP activity was enhanced in cocultures of C6 glial cells with E6CH neurons. Glial cells from aged mouse brain did not respond to coculturing with E6CH neurons. It appears from these findings that neuronal input enhances the differentiation of glioblastic cells to either astrocytic or oligodendrocytic expression, whereas it decreases the activity of committed astrocytes. In contrast, glial cells from aged mouse brain do not respond to neuronal input. Choline acetyltransferase (ChAT) activity, a marker for cholinergic neurons, was enhanced only when E6CH cultures were grown in conditioned medium (CM) from 2B23 glioblastic cells. In contrast, ChAT activity was markedly diminished when E6CH neurons were cocultured with MACH glial cells but not when grown in CM from MACH glial cells. Thus, humoral factors from immature glial cells appear to enhance cholinergic neuronal phenotypic expression whereas cell-cell membrane contacts with aged glial cells diminish cholinergic phenotypic expression. The findings present supportive evidence that neuron-glia interrelationships are age dependent.     

Calcium signaling in specialized glial cells

This article reviews calcium signaling in three specialized types of glial cells: Müller cells of the retina, Bergmann glial cells of the cerebellum, and radial glial cells of the developing cortex. Müller cells generate spontaneous and neuronal activity-evoked increases in Ca(2+). Neuron to Müller cell signaling is mediated by neuronal release of ATP and activation of glial P2Y receptors. Müller cells, in turn, modulate neuronal excitability and mediate vasomotor responses. Bergmann glial cells also generate spontaneous and activity-evoked Ca(2+) increases. Neuron to Bergmann glia signaling is mediated by neuronal release of nitric oxide, noradrenaline, and glutamate. In Bergmann glia, Ca(2+) increases control the structural and functional interactions between these cells and Purkinje cell synapses. In the ventricular zone of the developing cortex, radial glial cells generate spontaneous Ca(2+) increases that propagate as Ca(2+) waves through clusters of neighboring glial cells. These Ca(2+) increases control cell proliferation and neurogenesis.     

Müller cell differentiation in the zebrafish neural retina: evidence of distinct early and late stages in cell maturation

The vertebrate neural retina is mainly composed of cells of neuroectodermal origin. The primary cell types found in all vertebrate retinas are several categories of neurons and the archetypical retina glial cell the Müller cell. Although the neurons and the single glial cell type of the retina are specialized for very distinct functions, they all have a common developmental origin within the tissue. How the distinctions between cell types, in particular between neurons and glia, arise during embryonic development remains a central issue in neurobiology. In this report, we examine the genesis of Müller glial cells during zebrafish (Danio rerio) eye development. Particular emphasis is placed on the expression of the Müller cell maturation markers carbonic anhydrase and glutamine synthetase. In addition, we report that the HNK-1 monoclonal antibody, which identifies a particular glycoconjugate frequently found on cell surface recognition molecules, also identifies zebrafish retina Müller cells early in development. The expression patterns of these three markers clearly show that the Müller cells mature in stages: HNK-1 labeling and glutamine synthetase arise earlier than carbonic anhydrase expression. In addition, the embryonic zebrafish neural retina is characterized by the presence of amoeboid, carbonic anhydrase-positive microglial cells even before the genesis of retinal neuroectodermal glia. The stepwise maturation of the glia is likely to be indicative of an overall retinal maturational program in which cell differentiation and the expression of certain phenotype-defining gene products may be separately regulated.     

Differential modulation of glutamate metabolizing enzymes in mouse and chick cultured glial cells by insulin

The effect of physiological concentrations of insulin (2 and 20 ng/ml) on glutamine synthetase (GS) and glutamate dehydrogenase (GDH) activities were compared in mouse and chick glial cells in culture. Addition of insulin to serum-containing medium increased the level of GS and GDH activities in glial cells prepared from 14-15-day-old embryonic mice. A similar but less pronounced effect was observed with glia derived from newborn mouse brain. In absence of serum, addition of insulin had no effect on the tested enzymes. The effects of insulin on enzymatic activities of glial cells from 14-15-day-old embryonic chick brain hemispheres were, in contrast, quite different. A significant decrease of GS activity was induced by the hormone, only in the absence of serum. Conversely, the presence of serum enhanced an inhibitory effect of insulin toward chick GDH. The different effects of insulin and the different serum dependence observed for the mammalian and the avian model could reflect fundamental chemical differences between both species as indicated by immunoelectrophoretic analysis. However, it can be concluded that insulin may be a physiological factor regulating glial maturation and amino acid neurotransmitter metabolism in the central nervous system.     

Role of endothelins as mediators of injury-induced alterations of glial glutamate turnover

Astroglia terminate glutamatergic neurotransmission and prevent excitotoxic extracellular glutamate concentration by clearing synaptically released glutamate through the high-affinity, sodium-dependent glutamate transporters GLT-1 and GLAST. Many brain injures are associated with the disturbed expression of glial glutamate transporters and a subsequent increase of extracellular glutamate to neurotoxic levels. We have now followed up initial hints pointing to endothelins, a family of injury-regulated peptides, as mediators of this injury-induced loss of glial glutamate transporter expression. We observed that, in line with such a role, endothelins not only act as potent inhibitors of basal and exogenously (dbcAMP)-induced expression of GLT-1 in cortical astrocytes as shown before, but likewise inhibit expression of GLT-1 or GLAST in astrocytes cultured from the diencephalon, mesencephalon, cerebellum, and spinal cord. We further demonstrate that endothelins equally inhibit GLT-1 expression in cortical slice cultures, a culture system closely resembling the in vivo situation. Although brain injuries are usually associated with an increase in the expression of the glutamate-converting enzyme glutamine synthetase, cultured cortical astrocytes maintained with endothelins showed an almost complete loss of glutamine synthetase. Interestingly, the inhibitory effects of endothelins on the expression of glutamine synthetase, but not of glutamate transporters, was overridden by high extracellular glutamate, indicating that the primarily inhibitory action of endothelins on the various components of glial glutamate turnover dissociates in the injured brain.     

Neuroinflammation and regulation of glial glutamate uptake in neurological disorders

Oxidative stress, neuroinflammation, and excitotoxicity are frequently considered distinct but common hallmarks of several neurological disorders, including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease. Although neuron degeneration and death are the ultimate consequences of these pathological processes, it is now widely accepted that alterations in the function of surrounding glial cells are key features in the progression of these diseases. In response to alteration in their local environment, microglia, commonly considered the resident immune cells of the nervous parenchyma, become activated and release a variety of soluble factors. Among these, proinflammatory cytokines and free radicals actively participate in the degenerative insults. In addition, excitotoxic neuronal damage resulting from excessive glutamate is frequently associated with impaired handling of extracellular glutamate by gliotic astrocytes. Although several research projects have focused on the biochemical mechanisms of the regulation of glial glutamate transporters, a relationship between activation of microglia and modulation of astrocytic glutamate uptake is now suggested. The aim of this review is to summarize and discuss the data showing an influence of inflammatory mediators and related free radicals on the expression and activity of glial glutamate transporters.     

CNS region-specific regulation of glial glutamate transporter expression

The neuronal cell death associated with certain neurodegenerative disorders as well as acute brain injuries is in part due to the reduced expression of glial glutamate transporters and the subsequent accumulation of toxic extracellular glutamate concentrations. Extracellular factors previously found to potently stimulate the expression of the glial glutamate transporters, GLT-1/EAAT2 and GLAST/EAAT1, in astroglial cultures of rat cerebral hemispheres are PACAP, TGF alpha, and EGF. In the present study, we sought to determine whether similar stimulatory influences apply for astroglia from other areas of the central nervous system (CNS). Immunoblot and real-time RT-PCR analysis of striatal astroglial cultures maintained for 72 h with PACAP, TGF alpha, or EGF revealed a prominent increase in GLT-1 and GLAST expression. In apparent contrast, all factors completely failed to affect GLT-1 and GLAST expression in astroglial cultures from the cerebellum, mesencephalon, and spinal cord between 36 h and 7 days. This failure was not due to the absence of functional recognition or transduction machineries for the extracellular factors as suggested by the additional observations that cerebellar, mesencephalic and spinal cord glia were capable of responding to stimulation with PACAP, TGF alpha, or EGF for 10 min with activation of CREB. Moreover, dibutyryl cyclic AMP (dbcAMP) potently promoted GLT-1 and/or GLAST expression in mesencephalic, cerebellar and spinal cord glia, further indicating that extracellular factors regulate glial glutamate transporter expression throughout the CNS. Together these findings identify PACAP, TGF alpha and EGF as potent regulators of glutamate transporter expression in striatal glia. In addition, these findings provide evidence for a CNS region-specific regulation of glial glutamate transport.     

Unilateral optic nerve crush induces bilateral retinal glial cell proliferation

The post-injury responses of retinal ganglion cells elicit a number of glial reactions which have not been completely understood. The bilateral pattern of non-neuronal retinal cell proliferation was examined in association with the differential fates of unilaterally injured adult retinal ganglion cells by means of bromodeoxyuridine (BrdU) immunocytochemistry. Lateralization of the glioproliferative events was studied by analysing both the experimental and the uninjured contralateral as well as matched retinas of sham-operated animals. Control adult rat retina included very few BrdU-positive cells within the nerve fibre and ganglion cell layers; however, experimental retinas of degenerating groups exhibited statistically significantly higher densities of newborn cells in most layers. Clusters of labelled cells were found in the inner plexiform layer related to OX-42 staining, indicating their microglial nature. Indeed, double-labelling experiments, after short-term unilateral optic nerve crushing, identified proliferating retinal glial cells in vivo. Both types of glia, astroglial and microglial cells, exhibited BrdU-positive labelling in injured as well as uninjured experimental rat retinas. Moreover, microglial proliferating cells were also identified in explanted retinal pieces after 2 days in culture. Affected and contralateral retinas responded similarly to the unilateral experimental manipulations applied with respect to BrdU labelling. The acute glial responses observed suggest that bilateral glial proliferation might represent a common response related to degeneration events in both retinas, i.e. ipsi- and contralateral to the experimental injury.     

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.