Aspartate Uptake Research Articles

Acidic amino acid clearance from CSF in the neonatal versus adult rat using ventriculo-cisternal perfusion.

The acidic amino acids aspartate and glutamate are excitatory neurotransmitters in the CNS. The clearance of this group of amino acids from CSF of adult and neonatal (7-day-old) rats was investigated. Ventriculo-cisternal perfusions with 14C-amino acids and 3H-dextran were carried out for up to 90 min. Uptake of the amino acids by the whole brain was measured, and the loss to blood was calculated. 3H-Dextran was included in the perfusate for measurement of CSF secretion rate. After 90-min perfusion, both aspartate and glutamate showed a similar uptake into the whole brain, and this did not change with age (p>0.05). However, clearance from CSF was greater in the adult, as was entry into blood from CSF. Addition of 5 mM excess unlabelled amino acid resulted in reduction in the brain uptake of both 14C-amino acids in the adult rat. In the neonate, addition of aspartate also reduced brain aspartate uptake, whereas addition of glutamate increased brain neonatal [14C]glutamate uptake. The rate of CSF secretion was significantly greater in the adult, 1.26+/-0.18 microl x min(-1) x g(-1), than in the neonate, 0.62+/-0.08 microl x min(-1) x g(-1), and the turnover of CSF was greater in adults (p<0.01). In summary, both aspartate and glutamate showed greater clearances from CSF in the adult than the neonate. This clearance was found to be by carrier-mediated mechanisms.

Foreign metallothionein-I expression by transient transfection in MT-I and MT-II null astrocytes confers increased protection against acute methylmercury cytotoxicity

The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular Na 2 51 CrO 4 efflux and d-[2,3- 3 H ]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 μM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant ( p<0.01) increase in Na 2 51 CrO 4 efflux and a significant ( p<0.05) decrease in the initial rate (1 min) of d-[2,3- 3 H ]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly ( p<0.01) decreased the effect of MeHg on Na 2 51 CrO 4 efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on d-[2,3- 3 H ]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 μM) were indistinguishable. These results demonstrate that: (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg.

Differentiation of substrate and nonsubstrate inhibitors of the high-affinity, sodium-dependent glutamate transporters.

Within the mammalian central nervous system, the efficient removal of L-glutamate from the extracellular space by excitatory amino acid transporters (EAATs) has been postulated to contribute to signal termination, the recycling of transmitter, and the maintenance of L-glutamate at concentrations below those that are excitotoxic. The development of potent and selective inhibitors of the EAATs has contributed greatly to the understanding of the functional roles of these transporters. In the present study, we use a library of conformationally constrained glutamate analogs to address two key issues: the differentiation of substrates from nontransportable inhibitors and the comparison of the pharmacological profile of synaptosomal uptake with those of the individual EAAT clones. We demonstrate that the process of transporter-mediated heteroexchange can be exploited in synaptosomes to rapidly distinguish transportable from nontransportable inhibitors. Using this approach, we demonstrate that 2,4-methanopyrrolidine-2,4-dicarboxylate, cis-1-aminocyclobutane-1,3-dicarboxylate, and L-trans-2, 4-pyrrolidine dicarboxylate act as substrates for the rat forebrain synaptosomal glutamate uptake system. In contrast, L-anti-endo-3, 4-methanopyrrolidine-3,4-dicarboxylate, L-trans-2,3-pyrrolidine dicarboxylate, and dihydrokainate proved to be competitive inhibitors of D-[(3)H]aspartate uptake that exhibited little or no activity as substrates. When these same compounds were characterized for substrate activity by recording currents in voltage-clamped Xenopus laevis oocytes expressing the human transporter clones EAAT1, EAAT2, or EAAT3, it was found that the pharmacological profile of the synaptosomal system exhibited the greatest similarity with the EAAT2 subtype, a transporter believed to be expressed primarily on glial cells.

Characterization of Aspartate Transport Across the Symbiosome Membrane in Pea Root Nodules

Summary Aspartate transport was studied in symbiosome membrane (SM) vesicles, facing bacteroid-side-out, purified from pea (Pisum sativum L.) root nodules. An intravesicular alkaline pH gradient (ΔpH) and an intravesicular negative membrane potential (Δψ) were imposed to energize the vesicles. Energized vesicles were capable of accumulating aspartate from the extravesicular medium against a concentration gradient. The uptake of aspartate in SM vesicles was electrogenic and the transport capacity was influenced by both ΔpH and Δψ. Furthermore, vesicle uptake of aspartate had an extravesicular pH optimum at 6. The uptake of aspartate driven by the energization of SM vesicles was biphasic showing saturation kinetics in the range of 0.01 to 1 mmol/L aspartate. Two protein modifying reagents, p-chloromercuribenzenesulphonic acid and phenylglyoxal, inhibited the uptake of aspartate in energized vesicles. The data are consistent with H+/aspartate symport(s) transporting aspartate from the bacteroid side of the SM to the plant cytosol.

Impairment of excitatory amino acid transport in astroglial cells infected with the human immunodeficiency virus type 1.

Perturbation of astrocyte functions by HIV-1 infection may contribute to the pathogenesis of AIDS dementia complex (ADC). The present study investigated the possibility that astroglial transport of glutamate and aspartate, the major excitatory amino acids (EAAs) in the mammalian central nervous system (CNS), is altered by HIV-1 infection. Human U251 glioma cells were infected with the brain isolate SF162 of HIV-1. HIV-1 persisted in glial cells over several months. This nonproductive infection of glial cells was characterized by persistent expression of Nef over the time of the infection, and the transient presence of structural viral proteins, including the viral transmembrane glycoprotein gp41, which was detected during the initial 2 weeks following HIV-1 infection. The presence of gp41 in acutely HIV-1-infected glial cells coincided with a 36% decrease in D-[3H]aspartate uptake, owing to a reduction in the maximal transport capacity (vmax) for D-aspartate. The expression of typical astrocytic glutamate transporters EAAT1 and EAAT2 in U251 glioma cells was not altered by HIV-1 infection. To determine whether viral protein gp120, gp41, or Nef was involved in the impairment of EAA transport in acutely HIV-1-infected glial cells, effects of lentiviral lytic peptide type 1 (LLP-1) (corresponding to the carboxy terminus of gp41), recombinant SF2 gp120, and recombinant LAI Nef on D-[3H]aspartate uptake and the release of glutamate in glial cells were investigated. Only LLP-1 reduced D-[3H]aspartate uptake and facilitated the release of glutamate from glial cells in a concentration-dependent manner. These results suggest that the carboxy terminus of gp41 impairs EAA transport in glial cells, which may contribute to excitotoxic damage to neurons in HIV-1 infection of the CNS.

Aspartate and alanine movement across symbiotic membranes of soybean nodules

Aspartate, alanine and glutamate uptake into bacteroids and symbiosomes isolated from soybean nodules were measured. An aspartate transport mechanism was identified in free bacteroids, with an apparent K m of 27.5 μ m and a V max of 5.14 nmol mg −1 min −1. Inhibitor studies indicated that aspartate uptake was dependent on a proton motive force across the bacteroid membrane. Aspartate transport was competitively inhibited by glutamate with an apparent K i of 9.7 μ m. Alanine uptake into bacteroids also showed saturation kinetics with a K m of 12.2 μ m and V max of 3.5 nmol mg −1 min −1. Bacteroids loaded with 14Caspartate exchanged label for external cold aspartate. and glutamate. In contrast, there was no evidence for aspartate or alanine movement into or out of intact symbiosomes except by slow passive diffusion.

Intravenous Aspartate Infusion After a Coronary Operation: Effects on Myocardial Metabolism and Hemodynamic State

Background. In a previous study glutamate infusion after coronary artery bypass grafting was associated with beneficial effects on myocardial metabolism and myocardial performance. It has been claimed that aspartate is more important than glutamate for the recovery of myocardial metabolism after cardioplegic arrest. Therefore, the metabolic and hemodynamic effects of aspartate were studied after coronary artery bypass grafting. Methods. Fifty to 240 mL of a 0.1 mol/L aspartic acid solution was infused intravenously during 60 minutes in 10 patients early after coronary artery bypass grafting. Myocardial metabolism was studied using the coronary sinus catheter technique. Results. Aspartate infusion caused a significant increase in the arterial levels of both aspartate and glutamate. This was associated with a significant increase in myocardial uptake of aspartate and a decrease in myocardial uptake of glutamate. Myocardial exchange of other substrates remained unaffected. There were no changes in hemodynamic state except an increase of heart rate and pulmonary vascular resistance. Conclusions. Interactions with glutamate metabolism, compatible with competitive inhibition of myocardial glutamate uptake, which may have outweighed potential effects of aspartate, were observed. Recognition of these amino acid interactions is important as they are used together as additives in cardioplegic solutions.

Amyloid Protein Precursor Stimulates Excitatory Amino Acid Transport: IMPLICATIONS FOR ROLES IN NEUROPROTECTION AND PATHOGENESIS

Excitatory neurotransmitters such as glutamate are required for the normal functioning of the central nervous system but can trigger excitotoxic neuronal injury if allowed to accumulate to abnormally high levels. Their extracellular levels are controlled primarily by transmitter uptake into astrocytes. Here, we demonstrate that the amyloid protein precursor may participate in the regulation of this important process. The amyloid protein precursor has been well conserved through evolution, and a number of studies indicate that it may function as an endogenous excitoprotectant. However, the mechanisms underlying this neuroprotective capacity remain largely unknown. At moderate levels of expression, human amyloid protein precursors increased glutamate/aspartate uptake in brains of transgenic mice, with the 751-amino acid isoform showing greater potency than the 695-amino acid isoform. Cerebral glutamate/aspartate transporter protein levels were higher in transgenic mice than in non-transgenic controls, whereas transporter mRNA levels were unchanged. Amyloid protein precursor-dependent stimulation of aspartate uptake by cultured primary astrocytes was associated with increases in protein kinase A and C activity and could be blocked by inhibitors of these kinases. The stimulation of astroglial excitatory amino acid transport by amyloid protein precursors could protect the brain against excitotoxicity and may play an important role in neurotransmission.

Metabolic Changes in Giardia intestinalis during Differentiation

The oxygen uptake rate and metronidazole (MTZ) sensitivity in Giardia spp. cysts is greatly reduced from that in trophozoites. Thus, this project was undertaken to assess when in the encystation process these phenomena occur. Oxygen uptake rates approximately doubled (from approximately 4.9 to 8.3 microM O2 min(-1) 10(-6) cells) during the first 5 hr into encystation. This increase was followed by a marked decrease to 2.3 microM O2 min(-1) 10(-6) by 12 hr. By 50 hr into encystation, oxygen uptake was 0.7 microM O2 min(-1) 10(-6) cells. Glucose stimulated oxygen uptake by 89% in trophozoites but did not demonstrably stimulate oxygen uptake in cells after 12 hr into encystment. Deoxy-D-glucose uptake dropped by more than an order of magnitude in encysting cells compared to nonencysting cells. In contrast, aspartate uptake remained relatively constant regardless of whether cells were encysting or not. This suggests that there is a change in the parasite's ability to transport glucose during cyst formation; a similar change in the parasite's ability to transport aspartate was not observed after 40 hr into encystation. MTZ inhibited oxygen uptake by 77% in trophozoites, but there was no detectable inhibition of oxygen uptake 8 hr after trophozoites were transferred to encystation medium. We propose that this resistance to MTZ may be due to a change in metabolic flux away from the pyruvate ferredoxin oxidoreductase pathway. Oxygen uptake by noninduced cysts increased exponentially during the 30 min following the induction of excystation. Likewise, MTZ sensitivity returned within 15 min after the induction of excystation, and by 30 min into excystation full sensitivity had returned.

Anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge

Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of γ-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA.

Anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge

Mechanisms of the anaerobic uptake of glutamate and aspartate by enhanced biological phosphorus removal activated sludge were investigated. Sludge hydrolysate with hydrochloric acid was analyzed by reverse phase HPLC after pre column derivatization with dabsyl-chloride. The experimental results indicated that glutamate is accumulated as a polymer consisting of γ-aminobutyric acid and an unknown amino acid. On the other hand, aspartate was found to be deaminated and then accumulated as PHA.

Aspartate as a selective NMDA receptor agonist in cultured cells from the avian retina

Although glutamate is considered the natural neurotransmitter that mediates excitatory function in the CNS, other active natural compounds can also drive the functional activation of excitatory amino acid receptors (EAAR). L-aspartate is the most likely neurotransmitter to mimic the actions of glutamate. Here we show that L-aspartate promotes the release of GABA acting selectively on the NMDA receptor subtype. Retina cell cultures, when exposed to excitatory amino acids (EAA), release [ 3H] GABA previously incorporated by the cells. Both L-glutamate and L- and D-aspartate at 100 μM concentration, promote the release which can be mimicked by kainate and NMDA. While aspartate-induced release of [ 3H] GABA occurs in the presence of 1 mM Mg 2+, NMDA (100 μM) promotes the release only when Mg 2+ is omitted from the superfusing medium. However, in the absence of Mg 2+ the efficacy of 1- and d-aspartate (100 μM) to activate [ 3H] GABA release increases by a factor of 2 when compared to the release observed in the presence of 1 mM Mg 2+. NMDA and aspartate induced release of [ 3H] GABA is completely inhibited by 10 μM MK-801 and is not affected by CNQX (100 μM). In the presence of Mg 2+, aspartate-induced release of [ 3H] GABA is also completely inhibited by MK-801 (10 μM) and is not significantly affected by CNQX (100 μM). The [ 3H] GABA release induced by kainate (100 μM) is fully inhibited by CNQX (100 μM) and is not affected by MK-801 (10 μM). Our results indicate that in the retina, l-aspartate modulates its excitatory function on a set of GABAergic cells via the selective activation of NMDA receptors. The fact that L- and D-aspartate (but not D-glutamate) induce the release of GABA even in the presence of Mg 2+ suggests that the electrogenic uptake of aspartate is required to lower the affinity of the NMDA channel for Mg 2+. The observation that D-glutamate (200 μM), which is not taken up by the cells, activates the efflux of GABA only when Mg 2+ is omitted from the incubating medium, supports this possibility.

Rat adipose tissue amino acid metabolism in vivo as assessed by microdialysis and arteriovenous techniques.

In fed, anesthetized rats, microdialysis demonstrated a net release of glycerol, glutamine, serine, tyrosine, and taurine and a net uptake of glutamate, aspartate, glycine, and arginine across the inguinal adipose depot. However, the results also indicated excessive proteolysis associated with implantation of the microdialysis probe, and a novel arteriovenous difference technique was developed. Arteriovenous difference across the inguinal fat pat demonstrated a net uptake of glucose and a net release of lactate and glycerol. Starvation (48 h) resulted in higher rates of glycerol and lactate release with lower rates of glucose uptake. A net uptake of triacylglycerol was seen in starved-refed animals. Net glutamine, tyrosine, and taurine release was seen in fed and starved animals, but in starved-refed animals taurine and serine were the only amino acids showing significant release. No significant net uptake or release of ammonia, pyruvate, or alanine was observed. These experiments confirm that adipose tissue is a site of glutamine synthesis and suggest that the principal substrates are derived from intracellular proteolysis. The results also demonstrate the viability of an arteriovenous difference technique for the study of adipose tissue in the rat.

Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants.

In the present study we analyzed how oxidative stress conditions induced by ascorbate/ Fe2+ affect the excitatory amino acid (EAA) transport systems in cultured chick retina cells. The uptake of D-[3H]aspartate, which is transported by the same carrier as glutamate, was determined in control cells and in cells subjected to ascorbate/Fe2+. The uptake of this EAA was Na+ dependent and was inhibited by about 40% under oxidative stress conditions. To clarify the molecular mechanisms involved in the inhibition of D-[3H]aspartate uptake by ascorbate/Fe2+, we investigated the effect of vitamin E (Vit E), melatonin, reduced glutathione (GSH), and dithiothreitol (DTT) on the uptake of D-[3H]aspartate and on the extent of lipid peroxidation in control and in peroxidized cells. Preincubation with Vit E (100 microM) abolished lipid peroxidation, but had no significant effect on the inhibition of D-[3H]aspartate uptake evoked by ascorbate/Fe2+. Melatonin was more effective in reducing the formation of TBARS and conjugated dienes than in preventing the D-[3H]aspartate uptake inhibition evoked by the oxidant pair. Conversely, GSH (4 mM) and DTT (4 mM) completely prevented the inhibition of D-[3H]aspartate uptake in cells subjected to oxidative stress, but were without effect on the extent of peroxidation. Free fatty acids, such as arachidonic acid, seem not to be involved in reducing the activity of the D-[3H]aspartate uptake system, whereas the reduction of the Na+ electrochemical gradient that occurs under oxidative stress was in part involved in the reduction of D-[3H]aspartate uptake by the cells. The inhibition of D-[3H]aspartate uptake by ascorbate/Fe2+ persisted for at least 1 h, but could be partially reverted by disulfide reducing agents. It is concluded that oxidative stress causes long-lasting modifications of the glutamate/D-[3H]aspartate transport system (or systems), such as oxidation of protein sulfhydryl (SH) groups, which can be recovered by some antioxidants.

Evidence for increased cellular uptake of glutamate and aspartate in the rat hippocampus during kainic acid seizures

Using a newly developed technique, based on microdialysis, which allows cellular uptake of glutamate and aspartate to be studied in awake animals, we investigated uptake of glutamate and aspartate in the hippocampal formation of rats during limbic seizures induced by systemical administration of kainic acid (KA). With [14C]mannitol as an extracellular reference substance, the cellular extraction of the test substance [3H]D-aspartate was measured at different stages of seizure-activity. The results were compared to those obtained in a sham operated control group. During severe generalized clonic seizures, the extraction of [3H]D-aspartate was increased by 17%. The increase in uptake of [3H]D-aspartate was accompanied by a 24% increase in the extracellular level of aspartate, as obtained by conventional microdialysis. No significant changes were observed in the extracellular level of glutamate. The results indicate that during KA-induced seizures, uptake of glutamate and aspartate is increased, possibly aimed at maintaining the extracellular homeostasis of these two excitatory amino acids.

Effects of expressing E. coli threonine synthase in tobacco ( Nicotiana tabacum L.) suspension culture cells on free amino acid levels, aspartate pathway enzyme activities and uptake of aspartate into the cells

Summary The amino acids threonine and methionine are derived from aspartate in a multibranched biosynthetic pathway. In higher plants, the branchpoint enzymes, threonine synthase (TS; EC 4.2.99.2) and cystathionine γ-synthase (CγS; EC 4.2.99.9), which lead to threonine/isoleucine and methionine synthesis, respectively, compete against each other for pathway intermediates. In order to better understand the regulation and interplay between these competing pathways, the feedback-insensitive E. coli TS was constitutively expressed in tobacco ( Nicotiana tabacum L.) suspension cultured cells via Agrobacterium -mediated transformation. Expression of E. coli TS in tobacco cells resulted in a 7-fold increase in total TS activity and a 5.7-fold increase in free threonine levels. CγS activity increased 3.5 fold, apparendy to compensate for the heightened competition for the common pathway intermediate homoserine phosphate. Homoserine dehydrogenase and threonine-sensitive aspartate kinase activities were increased by almost 2 fold. Free aspartate was decreased by 55 % in the transformed cells, while free lysine and isoleucine levels were not significantly changed, indicating that threonine does not regulate its own synthesis by inhibiting an enzyme early in the pathway. Transformed cells had a markedly reduced ability to take up 14 [C] aspartate from the medium, suggesting that threonine may regulate its synthesis in vivo in part by limiting aspartate availability after Met, Lys, and lie pools are filled.

Glutamate receptor agonists up-regulate glutamate transporter GLAST in astrocytes.

Long-term treatment of astrocytes in primary culture with L-glutamate (0.1-3 mM) resulted in a dose-dependent increase in D-[3H]aspartate uptake. The effect was abolished by an antagonist of kainate/AMPA receptors, CNQX, and mimicked by kainate, but not by AMPA or tACPD. Both glutamate and kainate caused a dramatic up-regulation (82% and 69%, respectively) of GLAST, a predominant glutamate transporter in cultured astroglia, though the mRNA levels appeared unaffected. Long-term treatment of cultures with dBcAMP stimulated D-[3H]aspartate uptake as well as GLAST expression. Apart from glutamate, none of the agonists used was capable of increasing further the uptake capacity of the dBcAMP-treated astroglia. The glutamate receptor-dependent modulation of glutamate transport in astroglial cultures may represent a novel feedback regulatory mechanism for glutamate uptake in the brain.

Effect of diabetes on levels and uptake of putative amino acid neurotransmitters in rat retina and retinal pigment epithelium.

Free amino acid levels and high affinity uptake of glutamate, aspartate, gamma-aminobutyrate, glycine and taurine were studied in retina and retinal pigment epithelium of streptozotocin diabetic rats. Results show that experimental diabetes produces a generalized fall in the content of free amino acids in both retina and retinal pigment epithelium. With regard to the high affinity uptake, in the two tissues of diabetic animals showed decreased aspartate uptake, enhanced taurine and gamma-aminobutyrate uptake, whereas that of glycine and glutamate was unchanged. These results might suggest that diabetes causes alterations of specific amino acid transport systems and/or alterations of some cell populations.

Kinetics, energetics and specificity of a general amino acid transporter from the ectomycorrhizal fungus Paxillus involutus

The kinetics, energetics and specificity of a general amino acid transporter were studied in the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. The uptake of amino acids showed features characteristic of active transport. After correction for a non-mediated transport component, the kinetics of glutamate, glutamine, alanine and aspartate uptake measured over a wide concentration range followed the simple Michaelis-Menten saturation curves. The apparent K m derived from the Eadie-Hofstee plots ranged from 7 μM for alanine to 27 μM for glutamate. Dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone and NaN3 strongly inhibited amino acid uptake, whereas dicyclohexylcarbodiimide, vanadate and the ionophores monensin and nonactin had no effect on the uptake. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism for amino acid uptake by P. involutus. Competition studies indicated a broad substrate recognition by the uptake system, which resembles the general amino acid permease of yeast. Dixon plots of the inhibition of glutamate uptake by alanine, lysine and methionine sulfoximine showed that inhibitions were competitive. The physiological importance of this transporter for the exchange of nitrogenous compounds between fungal and host plant cells in ectomycorrhizal associations is discussed.

Serum affects the characteristics of excitatory amino acid-binding sites on Müller cells

The binding of [ 3H] l-aspartate to membranes obtained from primary cultures of chick retinal Müller cells (glia) was studied. Cells seeded in low-serum-containing medium (1%) and maintained in this condition showed an increased number of binding sites, from 1 to 5 days in vitro (DIV), when compared with controls cultured in medium containing 10% serum; these changes were not reversed by the addition of 10% serum after 48 h in vitro. Increased binding at this age was due to the expression of a low affinity binding system, competitively inhibited by the glutamate uptake blocker l-aspartate-β-hydroxamate, suggesting that high serum might inhibit the expression of uptake sites at precise maturation stages. Experiments showed the effect was due to a thermolabile serum component. The increase in binding sites is parallel in time to both an increase in aspartate uptake and the initiation of synaptogenesis in the whole retina. Our results suggest that the presence of serum at defined stages in retinal development, could result in the elevation of extracellular glutamate and the concomitant excitotoxic death of neuronal cells, due to a decreased glutamate uptake by glial cells.