Immunohistochemical Localization of GFAP and Glutamate Regulatory Proteins in Chick Retina and Their Levels of Expressions in Altered Photoperiods

Moderate to intense light is reported to damage the chick retina, which is cone dominated. Light damage alters neurotransmitter pools, such as those of glutamate. Glutamate level in the retina is regulated by glutamate–aspartate transporter (GLAST) and glutamine synthetase (GS). We examined immunolocalization patterns and the expression levels of both markers and of glial fibrillary acidic protein (GFAP, a marker of neuronal stress) in chick retina exposed to 2000 lux under 12-h light:12-h dark (12L:12D; normal photoperiod), 18L:6D (prolonged photoperiod), and 24L:0D (constant light) at post-hatch day 30. Retinal damage (increased death of photoreceptors and inner retinal neurons and Müller cell hypertrophy) and GFAP expression in Müller cells were maximal in 24L:0D condition compared to that seen in 12L:12D and 18L:6D conditions. GS was present in Müller cells and GLAST expressed in Müller cell processes and photoreceptor inner segments. GLAST expression was decreased in 24L:0D condition, and the expression levels between 12L:12D and 18L:6D, though increased marginally, were statistically insignificant. Similar was the case with GS expression that significantly decreased in 24L:0D condition. Our previous study with chicks exposed to 2000 lux reported increased retinal glutamate level in 24L:0D condition. The present results indicate that constant light induces decreased expressions of GLAST and GS, a condition that might aggravate glutamate-mediated neurotoxicity and delay neuroprotection in a cone-dominated retina.     

Are neuronal transporters relevant in retinal glutamate homeostasis?

Exposure of isolated retinas to 30 microM D-aspartate, which is a substrate for all high affinity glutamate transporters, for 30 min, resulted in the accumulation of such D-aspartate into Müller glial cells but not glutamatergic neurons as evinced by immunocytochemistry for D-aspartate. Further incubation of such loaded retinas in physiological media, in the absence of D-aspartate, resulted in the slow release of accumulated D-aspartate from the Müller cells and its accumulation into populations of photoreceptors and bipolar cells. This result indicates that after initial transport into Müller cells, reversal of direction of transport of D-aspartate, and thus by inference glutamate, by GLAST, readily occurs. D-aspartate released by Müller cells was strongly accumulated into cone photoreceptors which are known to express GLT-1, and into rod photoreceptors which we demonstrate here to express the retina specific glutamate transporter EAAT5 (excitatory amino transporter 5). Populations of glutamatergic bipolar cells, which express GLT-1 also exhibited avid uptake of D-aspartate. We conclude that the Müller cell glutamate transporter GLAST is responsible for most of the initial glutamate clearance in the retina after its release from neurones. However, some glutamate is also returned from Müller cells, to neurons expressing GLT-1 and EAAT5, albeit at a slow rate. These data suggest that the role of neuronal glutamate transporters in the retina may be to facilitate a slow process of recycling glutamate back from Müller cells to neurons after its initial clearance from perisynaptic regions by GLAST.     

Resveratrol Inhibits Diabetic-Induced Müller Cells Apoptosis through MicroRNA-29b/Specificity Protein 1 Pathway

The aim of this study was to evaluate the anti-apoptosis effects of resveratrol (RSV) on diabetic rats retinal Müller cells in vivo and in vitro and to further investigate the roles of microRNA-29b (miR-29b)/specificity protein 1 (SP1) in the anti-apoptosis mechanism of RSV. Retina was obtained from normal and diabetic rats with or without RSV (5 and 10 mg/kg/day) treatments at 1–7 months. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and Annexin V/PI staining were used to detect apoptosis. Immunofluorescence was used to assess distribution of SP1 in retina. MiR-29b and SP1 messenger RNA (mRNA) expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). SP1, Bax, and bcl-2 protein expression was evaluated by western blotting. Caspase-3 activity was detected by assay kit. Our study showed that the TUNEL-positive cells were mainly localized in the inner nuclear layer (INL) of retina and RSV administration effectively suppressed streptozotocin (STZ)-induced apoptosis of retinal cells in INL in vivo (P?<?0.001). Our study also showed that RSV administration effectively suppressed high glucose (HG)-induced retinal Müller cells’ apoptosis in vitro (P?<?0.001). Furthermore, our study revealed that the diabetes-induced downregulated expression of miR-29b and upregulated expression of SP1 could be rescued by RSV in vivo and in vitro (P?<?0.05). The anti-apoptosis effect and downregulated SP1 expression effect of RSV was prevented by miR-29b inhibitor (P?<?0.05). MiR-29b mimic increased the above-mentioned effects of RSV (P?<?0.001). These findings indicate that RSV is a potential therapeutic option for diabetic retinopathy (DR) and that miR-29b/SP1 pathway play roles in the anti-apoptosis mechanism of RSV.     

Physiological response of bovine subcommissural organ to endothelin 1 and bradykinin

Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system x_c ⁻ (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na⁺-dependent and –independent uptake of [³H] glutamate was assessed as a measure of GLAST and system x_c ⁻ function, respectively. Hyperglycemia did not alter the uptake of [³H] glutamate by GLAST or system x_c ⁻; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system x_c ⁻ activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in V_max, but not K_m. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system x_c ⁻. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system x_c ⁻ activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione. This work was supported by NIH R01 EY014560.     

BDNF regulates GLAST and glutamine synthetase in mouse retinal Müller cells

This study investigated whether brain-derived neurotrophic factor (BDNF) regulates the L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) in mouse retinal Müller cells (RMCs) under normal and hypoxic conditions. Mouse RMCs were treated with recombinant human BDNF (50, 75, 100, 125, or 150 ng/ml) for 24 h or underwent hypoxia induced by CoCl(2) (125 μM; 6, 12, 24, 48, or 72 h). An additional group underwent combined treatment with BDNF (100 ng/ml; 24, 48, 72, or 96 h) and CoCl(2) (125 μM/ml; 72 h). GLAST and GS mRNA and protein expression, L-[3,4-3H]-glutamic acid uptake, and apoptosis were assessed. BDNF dose-dependently up-regulated GLAST and GS mRNA and protein and increased glutamate uptake. Similarly, in early-stage CoCl(2)-induced hypoxia, GLAST and GS were up-regulated and glutamate uptake increased, but these decreased over time. BDNF also up-regulated GLAST and GS and increased glutamate uptake when RMCs under CoCl(2) induced hypoxic condition. However, BDNF treatment 24 h before CoCl(2) had no effect on GLAST or GS expression. CoCl(2) alone or combined with BDNF did not induce apoptosis. Hypoxia rapidly increased GLAST and GS expressions. This effect was transient, perhaps due to compensatory mechanisms that reduce GLAST and GS by 72 h. BDNF can up-regulate GLAST and GS and increase glutamate uptake during hypoxia, and these functions may underlie its neuroprotective effects.     

Thioredoxin 1 overexpression attenuated diabetes-induced endoplasmic reticulum stress in Müller cells via apoptosis signal-regulating kinase 1

As one of the common and serious chronic complications of diabetes mellitus (DM), the related mechanism of diabetic retinopathy (DR) has not been fully understood. Müller cell reactive gliosis is one of the early pathophysiological features of DR. Therefore, exploring the manner to reduce diabetes-induced Müller cell damage is essential to delay DR. Thioredoxin 1 (Trx1), one of the ubiquitous redox enzymes, plays a vital role in redox homeostasis via protein–protein interactions, including apoptosis signal-regulating kinase 1 (ASK1). Previous studies have shown that upregulation of Trx by some drugs can attenuate endoplasmic reticulum stress (ERS) in DR, but the related mechanism was unclear. In this study, we used DM mouse and high glucose (HG)-cultured human Müller cells as models to clarify the effect of Trx1 on ERS and the underlying mechanism. The data showed that the diabetes-induced Müller cell damage was increased significantly. Moreover, the expression of ERS and reactive gliosis was also upregulated in diabetes in vivo and in vitro. However, it was reversed after Trx1 overexpression. Besides, ERS-related protein expression, reactive gliosis, and apoptosis were decreased after transfection with ASK1 small-interfering RNA in stable Trx1 overexpression Müller cells after HG treatment. Taken together, Trx1 could protect Müller cells from diabetes-induced damage, and the underlying mechanism was related to inhibited ERS via ASK1.     

Limited Energy Supply in Müller Cells Alters Glutamate Uptake

The viability of retinal ganglion cells (RGC) is essential for the maintenance of visual function. RGC homeostasis is maintained by the surrounding retinal glial cells, the Müller cells, which buffer the extracellular concentration of neurotransmitters and provide the RGCs with energy. This study evaluates if glucose-deprivation of Müller cells interferes with their ability to remove glutamate from the extracellular space. The human Müller glial cell line, Moorfields/Institute of Ophthalmology-Müller 1, was used to study changes in glutamate uptake. Excitatory amino acid transporter (EAAT) proteins were up-regulated in glucose-deprived Müller cells and glutamate uptake was significantly increased in the absence of glucose. The present findings revealed an up-regulation of EAAT1 and EAAT2 in glucose-deprived Müller cells as well as an increased ability to take up glutamate. Hence, glucose deprivation may result in an increased ability to protect RGCs from glutamate-induced excitotoxicity, whereas malfunction of glutamate uptake in Müller cells may contribute to retinal neurodegeneration.     

高糖环境对Mu ller细胞谷氨酸转运体及谷氨酰胺合成酶表达的影响

目的：研究高糖环境对原代培养新生7天SD乳鼠视网膜Muller细胞谷氨酸转运合成系统的影响及其可能机制。方法：新生7天SD乳鼠视网膜Muller细胞原代培养并模拟高糖环境构建乳鼠视网膜muller细胞体外高糖环境模型。处理分为3组：对照组,高糖组,高糖＋白藜芦醇干预组。培养时间为24h,通过westernblot等检测方法,对照观察各组Muller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶（GS）的表达情况。结果：模拟高糖环境可以造成新生SD乳鼠视网膜Muller细胞谷氨酸转运体（GLAST）表达的降低（0．225foldVScontrol,P〈0．05）,并导致其表达的谷氨酰胺合成酶（GS）表达水平的显著降低（0．653foldVScontrol,P〈0．05）;而干预药物白藜芦醇作用后可明显逆转新生SD乳鼠Mu ller细胞谷氨酸转运体（GLAST）（1．133foldvSHGgroup,P〈0．05）、谷氨酰胺合成酶（GS）（1．720foldVSHGgroup,P〈0．05）等蛋白的表达水平。结论：模拟高糖环境可以影响视网膜M0ller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶的表达,其结局可能导致视神经细胞因谷氨酸堆积而导致的兴奋性毒性,白藜芦醇能提高Mcjller细胞谷氨酸转运体（GLAST）、谷氨酰胺合成酶表达,从而保护视神经细胞。     

Glutamine synthetase enhances the clearance of extracellular glutamate by the neural retina

Clearance of synaptic glutamate by glial cells is required for the normal function of excitatory synapses and for prevention of neurotoxicity. Although the regulatory role of glial glutamate transporters in glutamate clearance is well established, little is known about the influence of glial glutamate metabolism on this process. This study examines whether glutamine synthetase (GS), a glial-specific enzyme that amidates glutamate to glutamine, affects the uptake of glutamate. Retinal explants were incubated in the presence of [(14)C]glutamate and glutamate uptake was assessed by measurement of the amount of radioactively labeled molecules within the cells and the amount of [(14)C]glutamine released to the medium. An increase in GS expression in Müller glial cells, caused by induction of the endogenous gene, did not affect the amount of glutamate accumulated within the cells, but led to a dramatic increase in the amount of glutamine released. This increase, which was directly correlated with the level of GS expression, was dependent on the presence of external sodium ions, and could be completely abolished by methionine sulfoximine, a specific inhibitor of GS activity. Our results demonstrate that GS activity significantly influences the uptake of glutamate by the neural retina and suggest that this enzyme may represent an important target for neuroprotective strategies.     

Role of retinal glial cells in neurotransmitter uptake and metabolism

In addition to photoreceptors and neurons, glial cells (in particular Müller cells) contribute to the removal and metabolization of neurotransmitters in the neural retina. This review summarizes the present knowledge regarding the role of retinal glial cells in the uptake of glutamate, N-acetylaspartylglutamate, γ-aminobutyric acid, glycine, and d-serine, as well as the degradation and removal of purinergic receptor agonists. Some major pathways of glutamate metabolism in Müller cells are described; these pathways are involved in the glutamate–glutamine cycle of the retina, in the defense against oxidative and nitrosative stress via the production of glutathione, and in the production of substrates for the neuronal energy metabolism. In addition, the developmental regulation of the major glial glutamate transporter, GLAST, and of the glia-specific enzyme glutamine synthetase is described, as well as the importance of a malfunction and even reversal of glial glutamate transporters, and a downregulation of the glutamine synthetase, as pathogenic factors in different retinopathies.     

Dietary Taurine Supplementation Prevents Glial Alterations in Retina of Diabetic Rats

The preventive effect of dietary taurine supplementation on glial alterations in retina of streptozotocin-induced diabetic rats was examined in this study. Blood glucose content, content of taurine, glutamate and <gamma>-amino butyric acid (GABA) and expression of glial fibrillary acid protein (GFAP), vascular endothelial growth factor (VEGF), glutamate transporter (GLAST), glutamine synthetase (GS) and glutamate decarboxylase (GAD) in retina were determined in diabetic rats fed without or with 5% taurine in a controlled trial lasting 12 weeks, with normal rats fed without or with 5% taurine served as controls. Dietary taurine supplementation could not lower glucose concentration in blood (P > 0.05), but caused an elevation of taurine content and a decline in levels of glutamate and GABA in retina of diabetic rats (P < 0.05). The content of GABA in normal control group was not altered by taurine supplementation. With supplementation of taurine in diet, lower expression of GFAP and VEGF while higher expression of GLAST, GS and GAD in retina of diabetic rats were determinated by RT-PCR, Western-blotting and immunofluorescence (P < 0.05). GFAP, VEGF, GLAST, GS and GAD expressions in normal controls were not altered by taurine treatment. This may have prospective implications of using taurine to treat complications in diabetic retinopathy.     

Endothelins Inhibit Osmotic Swelling of Rat Retinal Glial and Bipolar Cells by Activation of Growth Factor Signaling

Water accumulation in retinal glial (Müller) and neuronal cells resulting in cellular swelling contributes to the development of retinal edema and neurodegeneration. Here, we show that endothelin-1 (ET-1) dose-dependently inhibits the hypoosmotic swelling of Müller cells in freshly isolated retinal slices of control and diabetic rats, with a maximal inhibition at 100 nM. Osmotic Müller cell swelling was also inhibited by ET-2. The effect of ET-1 was mediated by activation of ET_A and ET_B receptors resulting in transactivation of metabotropic glutamate receptors, purinergic P2Y₁, and adenosine A₁ receptors. ET-1 (but not ET-2) also inhibited the osmotic swelling of bipolar cells in retinal slices, but failed to inhibit the swelling of freshly isolated bipolar cells. The inhibitory effect of ET-1 on the bipolar cell swelling in retinal slices was abrogated by inhibitors of the FGF receptor kinase (PD173074) and of TGF-β1 superfamily activin receptor-like kinase receptors (SB431542), respectively. Both Müller and bipolar cells displayed immunoreactivities of ET_A and ET_B receptor proteins. The data may suggest that neuroprotective effects of ETs in the retina are in part mediated by prevention of the cytotoxic swelling of retinal glial and bipolar cells. ET-1 acts directly on Müller cells, while the inhibitory effect of ET-1 on bipolar cell swelling is indirectly mediated, via stimulation of the release of growth factors like bFGF and TGF-β1 from Müller cells.     

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.     

Antagonists of protein kinase C inhibit rat retinal glutamate transport activity in situ

Neuronal and glial high‐affinity transporters regulate extracellular glutamate concentration, thereby terminating synaptic transmission and preventing neuronal excitotoxicity. Glutamate transporter activity has been shown to be modulated by protein kinase C (PKC) in cell culture. This is the first study to demonstrate such modulation in situ, by following the fate of the non‐metabolisable glutamate transporter substrate, d ‐aspartate. In the rat retina, pan‐isoform PKC inhibition with chelerythrine suppressed glutamate uptake by GLAST (glutamate/aspartate transporter), the dominant excitatory amino acid transporter localized to the glial Müller cells. This effect was mimicked by rottlerin but not by Gö6976, suggesting the involvement of the PKCδ isoform, but not PKCα, β or γ. Western blotting and immunohistochemical labeling revealed that the suppression of glutamate transport was not due to a change in transporter expression. Inhibition of PKCδ selectively suppressed GLAST but not neuronal glutamate transporter activity. These data suggest that the targeting of specific glutamate transporters with isoform‐specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis, e.g. glaucoma and amyotrophic lateral sclerosis.     

Temporal and spatial expression of CCN3 during retina development

NOV/CCN3 is one of the founding members of the CCN (Cyr61 CTGF NOV) family. In the avian retina, CCN3 expression is mostly located within the central region of the inner nuclear layer. As retinal development progresses and this retinal layer differentiates and matures, CCN3 expression forms a dorsal–ventral and a central–peripheral gradient. CCN3 is produced by two glial cell types, peripapillary cells and Müller cells, as well as by horizontal, amacrine, and bipolar interneurons. In retinal neurons and Müller cell cultures, CCN3 expression is induced by activated BMP signaling, whereas Notch signaling decreases CCN3 mRNA and protein levels in Müller cells and has no effect in retinal neurons. In Müller cells, the CCN3 expression detected may thus result from a balance between the Notch and BMP signaling pathways. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

High-affinity glutamate transporter GLAST/EAAT1 regulates cell surface expression of glutamine/neutral amino acid transporter ASCT2 in human fetal astrocytes

Neutral amino acid transporter ASCT2, together with high-affinity glutamate transporters, belongs to the SLC1 gene family of Na(+)-dependent solute carriers and is one of the major transporters of glutamine in cultured astrocytes. Besides glutamine and other high-affinity substrates--alanine, serine, cysteine or threonine, ASCT2 can also translocate protonated glutamate. The present study elucidated substrate-dependent trafficking of ASCT2 in differentiated primary cultures of human fetal astrocytes. The differentiation induced by 8-bromo-cAMP caused dramatic up-regulation of two co-localized and functionally linked astroglial proteins--glutamate transporter GLAST, that is the only high-affinity router of glutamate into cultured astrocytes, and glutamine synthetase (GS), a cytosolic enzyme that converts at least a part of the arriving glutamate into glutamine. In order to distinguish individual intracellular effects of these two substrates on ASCT2, in some cultures glutamine synthetase was effectively knocked down using siRNA silencing technique. In control conditions, regardless of GS levels, almost the entire ASCT2 immunoreactivity was restricted to the cytosol. Both glutamine and alanine, though to different extents, induced partial redistribution of ASCT2 from the cytosolic compartment to the plasma membrane. However, in cultures with high GS expression, micromolar concentrations of glutamate exhibited more pronounced effect on ASCT2 trafficking than the preferred substrates of this carrier. In contrast, glutamate had no effect on ASCT2 distribution in cultures devoid of GS. D-Aspartate, a metabolically inert substrate effectively transported by GLAST, had no effect in any cell culture utilized. It seems that intracellular glutamine produced by GS from glutamate that, in turn, is supplied by GLAST, is a more potent inducer of ASCT2 trafficking to the cell surface than the ASCT2-mediated translocation of extracellular substrates. At lower pH values (6.2-6.7), the cell surface pool of ASCT2 was significantly larger than at physiological pH. In addition, high concentrations of glutamate, independently from GLAST or glutamate receptor activation, induced further arrival of ASCT2 to the plasma membrane. The pH-dependent functional activation of ASCT2 and the ASCT2-mediated glutamate uptake may play important roles during ischemic acidosis or synaptic activity-induced local acidification.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE AND DL-α-AMINOADIPIC ACID ON RAT RETINA: CHANGES IN HIGH AFFINITY UPTAKE OF PUTATIVE TRANSMITTERS

Subcutaneous administration of high doses of sodium glutamate to new born rats was used to destroy retinal interneurons and ganglion cells. Such treatment was accompanied by 90% reduction in the high affinity uptake of choline, 60–70% reductions in the uptakes of GABA, diamino-n-butyric acid and glycine and 30–40% reductions in the uptakes of asparatate and glutamate measured on retinal homogenates from 30-day-old rats. The high affinity uptakes of β-alanine and taurine were unchanged. Preincubation of retinal homogenates with 1 mM β-alanine or 100 μM diamino-n-butyric acid severely reduced the high affinity GABA uptake in control and experimental animals. In intact retinae, however, the glutamate treatment increased the high affinity uptake of β-alanine by 70%, whereas that of diamino-n-butyric acid was reduced by 40% and the high affinity uptakes of GABA and glutamate were unchanged. Four hours after injection of the gliotoxic compound DL-α-aminoadipic acid into the vitreous body of 30-day-old rats, the Müller cells could no longer be identified. This lesion was accompanied by 55% reduction in the high affinity uptake of β-alanine and 25% reduction in the uptakes of GABA and glutamate on intact retinae. The high affinity uptakes of diamino-n-butyric acid, choline and the enzyme activities of choline acetyltransferase and glutamate decarboxylase were unchanged under these conditions. After 24 h, however, the Müller cells could be recognized again, and the β-alanine uptake had normalized.     

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Müller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min. Following euthanasia, the retina was processed for D-aspartate, GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Müller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal, its ability to transport D-aspartate into Müller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease.