Lower Glutamate Levels Research Articles

Gamma-hydroxybutyrate accumulation in Arabidopsis and tobacco plants is a general response to abiotic stress: putative regulation by redox balance and glyoxylate reductase isoforms.

Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol are probably crucial in maintaining plant health during stress. Succinic semialdehyde (SSA) is a mitochondrially-generated intermediate in the metabolism of γ-aminobutyrate (GABA), which accumulates in response to a variety of biotic and abiotic stresses. SSA can be reduced to γ-hydroxybutyrate (GHB) under oxygen deficiency and high light conditions. Recent evidence indicates that distinct cytosolic and plastidial glyoxylate reductase isoforms from Arabidopsis (designated hereinafter as AtGR1 and AtGR2, respectively) catalyse the in vitro conversion of SSA to GHB, as well as glyoxylate to glycolate, via NADPH-dependent reactions. In the present report, the responses of GHB and related amino acids, as well as NADP+ and NADPH, were monitored in leaves from Arabidopsis or tobacco plants subjected to various abiotic stresses (i.e. Arabidopsis during exposure to salinity, drought, submergence, cold, or heat; tobacco during exposure to, and recovery from, submergence). Time-course experiments revealed that GHB accumulated in both Arabidopsis and tobacco plants subjected to stress, and that this accumulation was generally accompanied by higher GABA and alanine levels, higher NADPH/NADP+ ratio, and lower glutamate levels. Furthermore, the analysis of gene expression in Arabidopsis revealed that the relative abundance of GR1 (salinity, drought, submergence, cold, and heat) and GR2 (cold and heat) transcripts was enhanced by the stress tested. Thus, GHB accumulation in plants is a general response to abiotic stress and appears to be regulated by both biochemical and transcriptional processes.

Presynaptic transporter-mediated release of glutamate evoked by the protonophore FCCP increases under altered gravity conditions

High-affinity Na +-dependent glutamate transporters of the plasma membrane mediate the glutamate uptake into neurons, and thus maintain low levels of extracellular glutamate in the synaptic cleft. The study focused on the release of glutamate by reversal of Na +-dependent glutamate transporters from rat brain nerve terminals (synaptosomes) under conditions of centrifuge-induced hypergravity. Flow cytometric analysis revealed similarity in the size and cytoplasmic granularity between synaptosomal preparations obtained from control and G-loaded animals (10 G, 1 h). The release of cytosolic l-[ 14C]glutamate from synaptosomes was evaluated using the protonophore FCCP, which dissipated synaptic vesicle proton gradient, thus synaptic vesicles were not able to keep glutamate inside and the latter enriched cytosol. FCCP per se induced the greater release of l-[ 14C]glutamate in hypergravity as compared to control (4.8 ± 1.0% and 8.0 ± 1.0% of total label). Exocytotic release of l-[ 14C]glutamate evoked by depolarization was reduced down to zero after FCCP application under both conditions studied. Depolarization stimulated release of cytosolic l-[ 14C]glutamate from synaptosomes preliminary treated with FCCP was considerably increased from 27.0 ± 2.2% of total label in control to 35.0 ± 2.3% in hypergravity. Non-transportable inhibitor of glutamate transporter dl-threo-β-benzyloxyaspartate was found to significantly inhibit high-KCl and FCCP-stimulated release of l-[ 14C]glutamate, confirming the release by reversal of glutamate transporters. The enhancement of transporter-mediated release of glutamate in hypergravity was found to result at least partially from the inhibition of the activity of Na/K-ATPase in the plasma membrane of synaptosomes. We suggested that hypergravity-induced alteration in transporter-mediated release of glutamate indicated hypoxic injury of neurons.

Astrocytic glutamate targets NMDA receptors

Astrocytic glutamate targets NMDA receptors

TBOA-sensitive uptake limits glutamate penetration into brain slices to a few micrometers

Removal of neurotransmitter from the extracellular space is crucial for normal functioning of the central nervous system. In this study, we have used high-affinity metabotropic glutamate receptors (mGluRs) expressed by hippocampal CA1 pyramidal cells to test how far bath-applied glutamate penetrates into slice tissue before being removed by uptake mechanisms. Activation of group I mGluRs by 100 microM DHPG produced an inward current of -48+/-10pA (I(mGluR)), which was blocked by application of group I mGluR antagonists. In contrast, bath application of 100 microM glutamate in the presence of a ionotropic glutamate receptor antagonist and TTX did not activate I(mGluR) in CA1 cells patch-clamped at a depth of approximately 30 microm. Similarly, sole inhibition of glutamate transporters by the broad-spectrum glutamate transporter antagonist TBOA did not induce I(mGluR) under the same conditions. Only if glutamate was co-applied with TBOA an I(mGluR) of -39+/-8pA was recorded which was also blocked by group I antagonists. The data suggest that TBOA-sensitive uptake mechanisms are able to maintain a steep concentration gradient of glutamate to such a degree that a CA1 neuron at a depth of 30 microm is exposed to low extracellular glutamate levels that are not sufficient to induce a detectable activation of group I mGluRs (< 2 microM).

The effects of doxycycline administration on amino acid neurotransmitters in an animal model of neonatal hypoxia-ischemia

Neonatal hypoxia-ischemia (HI) is a major contributor to many neurological, psychiatric and behavioral disorders. Previous studies in our laboratory have shown that a one-time dose of doxycycline (DOXY), even when given 3 h after HI insult, was neuroprotective and significantly reduced microglial activation and cleaved caspase-3 protein expression in the immature brain. In light of these data, the goal of this study was to investigate the effects of DOXY administration on amino acid neurotransmitters. Post-natal-day 7 rats received DOXY (10 mg/kg) or vehicle (VEH) concomitant with the onset of HI, and were euthanized 30 min, 1, 2 or 4 h post-HI ( n ≥ 6). Extracted brains were either immediately dissected for frontal cortex, striatum and hippocampal regions, or removed in their entirety and flash frozen in isopentane for histological analyses. Dissected regions were homogenized and aliquots were prepared for high performance liquid chromatography (HPLC) analyses of amino acid levels and brain levels of DOXY. HPLC extraction revealed that systemic administration of DOXY resulted in mean drug levels of 867.1 ± 376.1 ng/g of brain tissue. Histological analyses revealed microglial activation, caspase-3 activation and neuronal degeneration consistent with a mild injury in the regions most vulnerable to HI. We found that HI caused significant, time-dependent, regional changes in brain amino acids including glutamate, GABA, alanine, aspartate, asparagine, serine, glutamine, glycine and taurine. HI significantly increased glutamate levels in the hippocampus (HI + VEH = 15.8 ± 3.1 ng/μg versus control = 11.8 ± 1.4 ng/μg protein) 4 h post-HI ( p < 0.05). Pups treated with DOXY had lower glutamate levels (13.1 ± 2.4 ng/μg) when compared to VEH-treated pups (15.8 ± 3.1 ng/μg), however these values failed to reach significance. In addition, DOXY-treated pups had significantly lower alanine (HI + VEH = 1.1 ± 0.2 ng/μg versus HI + DOXY = 0.5 + 0.1 ng/μg) and serine (HI + VEH = 1.4 ± 0.4 ng/μg versus HI + DOXY = 0.7 + 0.1 ng/μg) levels in the hippocampus, 4 h post-HI. Similar normalizations and significant reductions in alanine and serine were seen in the cortex and striatum. These results show that in addition to its previously reported and well-documented anti-inflammatory and anti-apoptotic properties, DOXY has significant effects on amino acid neurotransmitters.

Hypoxia—Ischemia Preferentially Triggers Glutamate Depletion from Oligodendroglia and Axons in Perinatal Cerebral White Matter

Ischemia is implicated in periventricular white matter injury (PWMI), a lesion associated with cerebral palsy. PWMI features selective damage to early cells of the oligodendrocyte lineage, a phenomenon associated with glutamate receptor activation. We have investigated the distribution of glutamate in rat periventricular white matter at post-natal day 7. Immuno-electron microcopy was used to identify O4(+) oligodendroglia in control rats, and a similar approach was employed to stain glutamate in these cells before and after 90 mins of hypoxia-ischemia. This relatively brief period of hypoxia-ischemia produced mild cell injury, corresponding to the early stages of PWMI. Glutamate-like reactivity was higher in oligodendrocytes than in other cell types (2.13+/-0.25 counts/microm(2)), and declined significantly during hypoxia-ischemia (0.93+/-0.15 counts/microm(2): P<0.001). Astrocytes had lower glutamate levels (0.7+/-0.07 counts/microm(2)), and showed a relatively small decline during hypoxia-ischemia. Axonal regions contained high levels of glutamate (1.84+/-0.20 counts/microm(2)), much of which was lost during hypoxia-ischemia (0.72+/-0.20 counts/microm(2): P>0.001). These findings suggest that oligodendroglia and axons are the major source of extracellular glutamate in developing white matter during hypoxia-ischemia, and that astrocytes fail to accumulate the glutamate lost from these sources. We also examined glutamate levels in the choroid plexus. Control glutamate levels were high in both choroid epithelial (1.90+/-0.20 counts/microm(2)), and ependymal cells (2.20+/-0.28 counts/microm(2)), and hypoxia-ischemia produced a large fall in ependymal glutamate (0.97+/-0.08 counts/microm(2): P>0.001). The ependymal cells were damaged by the insult and represent a further potential source of glutamate during ischemia.

Modification of Epileptiform Discharges in Neocortical Neurons Following Glutamate Uptake Inhibition

Sodium-dependent high-affinity glutamate transporters regulate synaptic glutamate levels to maintain low ambient levels of glutamate and prevent excitotoxicity. Most studies using pharmacological inhibition of glutamate transport to examine the involvement of glutamate transporters in regulating synaptic activity have examined small synaptic currents. Using in vitro brain slices, we investigated the effects of uptake inhibition on two types of epileptiform activity, bicuculline-induced paroxysmal activity, and epileptiform responses in the freeze-lesion epilepsy model. In layer II/III pyramidal cells of the prefrontal cortex, inhibiting uptake with low concentrations of DL-threo-ss-benzyloxyaspartic acid (TBOA) (20 or 30 microM) prolonged bicuculline-induced epileptiform activity. At higher concentrations, TBOA (150 or 300 microM) caused a transient enhancement of epileptiform discharges that was followed by a decrease. In the freeze-lesion model, inhibiting uptake also increased the amplitude and response area of evoked activity. The prolongation of epileptiform activity exhibited by the inhibition of glutamate uptake (TBOA 20 or 30 microM) is attributed to an increase in the level of glutamate extracellularly during uptake blockade, resulting in sustained activation of glutamate receptors. The decrease in epileptiform activity at higher TBOA concentration could be due to glutamate receptor desensitization or loss of excitability due to a depolarization block. The present results suggest that decreases in glutamate uptake can be proconvulsant in the two models of epilepsy examined.

Mental retardation and hypotonia seen in the knock out mouse for Canavan disease is not due to succinate semialdehyde dehydrogenase deficiency

Canavan disease (CD) is an autosomal recessive disorder caused by aspartoacylase deficiency leading to accumulation of N-acetylaspartic acid and spongy degeneration of the brain. The mouse model for CD showed low levels of glutamate and γ-aminobutyric acid (GABA) in the brain. Whether the low levels of glutamate and GABA observed in the CD mouse brain lead to abnormal production of glutamate-GABA associated enzymes and resulting succinate production is not obvious. While glutamate dehydrogenase and α-ketoglutarate dehydrogenase complex activities are lower in the cerebellum and brain stem of the CD mouse, alanine aminotransferase and succinate semialdehyde dehydrogenase (SSADH) activities and succinate level are similar to the levels observed in the wild type. Deficiency of SSADH has been suggested to be associated with mental retardation and hypotonia, similar to the clinical features of CD. The normal SSADH activity in the CD mouse brain suggests that mental retardation and hypotonia seen in the CD mouse is not due to SSADH activity and if documented also in patients with CD.

Cloning, transport properties, and differential localization of two splice variants of GLT-1 in the rat CNS: implications for CNS glutamate homeostasis.

At least two splice variants of GLT-1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT-1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT-1, namely GLT-1alpha. We demonstrate that GLT-1alpha is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT-1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT-1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT-1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT-1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

Canavan disease: a monogenic trait with complex genomic interaction

Canavan disease (CD) is an inherited leukodystrophy, caused by aspartoacylase (ASPA) deficiency, and accumulation of N-acetylaspartic acid (NAA) in the brain. The gene for ASPA has been cloned and more than 40 mutations have been described, with two founder mutations among Ashkenazi Jewish patients. Screening of Ashkenazi Jews for these two common mutations revealed a high carrier frequency, approximately 1/40, so that programs for carrier testing are currently in practice. The enzyme deficiency in CD interferes with the normal hydrolysis of NAA, which results in disruption of myelin and spongy degeneration of the white matter of the brain. The clinical features of the disease are macrocephaly, head lag, progressive severe mental retardation, and hypotonia in early life, which later changes to spasticity. A knockout mouse for CD has been generated, and used to study the pathophysiological basis for CD. Findings from the knockout mouse indicate that this monogenic trait leads to a series of genomic interaction in the brain. Changes include low levels of glutamate and GABA. Microarray expression analysis showed low level of expression of GABA-A receptor (GABRA6) and glutamate transporter (EAAT4). The gene Spi2, a gene involved in apoptosis and cell death, showed high level of expression. Such complexity of gene interaction results in the phenotype, the proteome, with spongy degeneration of the brain and neurological impairment of the mouse, similar to the human counterpart. Aspartoacylase gene transfer trial in the mouse brain using adenoassociated virus (AAV) as a vector are encouraging showing improved myelination and decrease in spongy degeneration in the area of the injection and also beyond that site.

Synaptic Activation of Presynaptic Glutamate Transporter Currents in Nerve Terminals

Glutamate uptake by high-affinity transporters is responsible for limiting the activation of postsynaptic receptors and maintaining low levels of ambient glutamate. The reuptake process generates membrane currents, which can be activated by synaptically released glutamate in glial cells and some postsynaptic neurons. However, less is known about presynaptic transporter currents because the small size of synaptic boutons precludes direct recordings. Here, we have recorded from two giant nerve terminals: bipolar cell synaptic terminals in goldfish retina and the calyx of Held in rat auditory brainstem. Exocytosis was evoked by brief depolarizations and measured as an increase in membrane capacitance. In isolated bipolar cell terminals, exocytosis was associated with an anion (NO3- or Cl-) current. The current peaked 2.8 msec after the start of the depolarization and decayed with a mean time constant of 8.5 msec. It was inhibited by the nontransportable glutamate transporter antagonist sc-threo-beta-benzyloxyaspartate (TBOA) but was insensitive to the GLT1/EAAT2 subtype-selective antagonist dihydrokainate and was affected by extracellular pH buffering. A TBOA-sensitive anion current was also evoked by application of exogenous glutamate to bipolar cell terminals. The large single-channel conductance, derived from noise analysis, and previous immunolocalization studies suggest that synaptically released glutamate activates EAAT5-type transporters in bipolar cell terminals. In contrast, neither exocytosis nor exogenous glutamate evoked a transporter current in the calyx of Held. Glutamate transporter currents with rapid kinetics are therefore identified and characterized in bipolar cell terminals, providing a valuable system for investigating the function and modulation of presynaptic glutamate transporters.

Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120

l-Glutamate is the major excitatory neurotransmitter in the brain. Astrocytes maintain low levels of synaptic glutamate by high-affinity uptake and defects in this function may lead to neuronal cell death by excitotoxicity. We tested the effects of HIV-1 and its envelope glycoprotein gp120 upon glutamate uptake and expression of glutamate transporters EAAT1 and EAAT2 in fetal human astrocytes in vitro. Astrocytes isolated from fetal tissues between 16 and 19 weeks of gestation expressed EAAT1 and EAAT2 RNA and proteins as detected by Northern blot analysis and immunoblotting, respectively, and the cells were capable of specific glutamate uptake. Exposure of astrocytes to HIV-1 or gp120 significantly impaired glutamate uptake by the cells, with maximum inhibition within 6 h, followed by gradual decline during 3 days of observation. HIV-1-infected cells showed a 59% reduction in V max for glutamate transport, indicating a reduction in the number of active transporter sites on the cell surface. Impaired glutamate transport after HIV-1 infection or gp120 exposure correlated with a 40–70% decline in steady-state levels of EAAT2 RNA and protein. EAAT1 RNA and protein levels were less affected. Treatment of astrocytes with tumor necrosis factor-α (TNF-α) decreased the expression of both EAAT1 and EAAT2, but neither HIV-1 nor gp120 were found to induce TNF-α production by astrocytes. These findings demonstrate that HIV-1 and gp120 induce transcriptional downmodulation of the EAAT2 transporter gene in human astrocytes and coordinately attenuate glutamate transport by the cells. Reduction of the ability of HIV-1-infected astrocytes to take up glutamate may contribute to the development of neurological disease.

Expression of glutamate transporter, GABRA6, serine proteinase inhibitor 2 and low levels of glutamate and GABA in the brain of knock-out mouse for Canavan disease

Canavan disease (CD) is an autosomal recessive leukodystrophy characterized by spongy degeneration of the brain. The clinical features of CD are hypotonia, megalencephaly, and mental retardation leading to early death. While aspartoacylase (ASPA) activity increases with age in the wild type mouse brain, there is no ASPA activity in the CD mouse brain. So far ASPA deficiency and elevated NAA have been ascribed with the CD. Other factors affecting the brain that result from ASPA deficiency may lead pathophysiology of CD. The NMR spectra and amino acid analysis showed lower levels of glutamate and γ-aminobutyric acid in the CD mouse brain compared to the wild type. Microarray gene expression on CD mouse brain showed glutamate transporter-EAAT4 and γ-aminobutyric acid-A receptor, subunit α6 (GABRA6) were lower 9.7- and 119.1-fold, respectively. Serine proteinase inhibitor 2 (Spi2) was 29.9-fold higher in the CD mouse brain compared to the wild type. The decrease of GABRA6 and high expression of Spi2 in CD mouse brain were also confirmed by real-time RT-PCR. This first report showing abnormal expression of EAAT4, GABRA6, Spi2 combined with lower levels of glutamate and GABA are likely to be associated with the pathophysiology of CD.

Activation of NR1a/NR2B receptors by monocyte-derived macrophage secretory products: implications for human immunodeficiency virus type one-associated dementia

The final pathways for neuronal injury in human immunodeficiency virus type one (HIV-1)-associated dementia (HAD) were investigated in Xenopus oocytes expressing recombinant NR1a/NR2B N-methyl- d-aspartate (NMDA) receptors exposed to secretory products from HIV-infected macrophages. Pressure ejection of HIV-1-infected and CD40 ligand-stimulated human monocyte-derived macrophage (MDM) fluids produced inward currents in oocytes expressing NR1a/NR2B (30.2±5.1 nA, n=42, mean±SE), but not in uninjected cells. In contrast, control (uninfected MDM) fluids induced currents of 4.5±0.5 nA ( n=17). Infected or stimulated MDM without virus showed intermediate responses. The induced currents were MDM fluid dose-dependant and blocked by the NMDA receptor antagonist 2-amino-5-phosphnovalerate (50 μM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM). Although low levels of glutamate were detected in the culture fluids, the addition of l-glutamate decarboxylase to the MDM did not significantly change the level of induced inward currents. Our experiments demonstrate that secretory factors from HIV-1-infected MDM activate NMDA receptors NR1a/NR2B and may contribute to neuronal demise during HAD.

Influence of cytosolic and mitochondrial Ca 2+, ATP, mitochondrial membrane potential, and calpain activity on the mechanism of neuron death induced by 3-nitropropionic acid

3-Nitropropionic acid (3NP), an irreversible inhibitor of succinate dehydrogenase, induces both rapid necrotic and slow apoptotic death in rat hippocampal neurons. Low levels of extracellular glutamate (10 μM) shift the 3NP-induced cell death mechanism to necrosis, while NMDA receptor blockade results in predominantly apoptotic death. In this study, we examined the 3NP-induced alterations in free cytosolic and mitochondrial calcium levels, ATP levels, mitochondrial membrane potential, and calpain and caspase activity, under conditions resulting in the activation of apoptotic and necrotic pathways. In the presence of 10 μM glutamate, 3NP administration resulted in a massive elevation in [Ca 2+] c and [Ca 2+] m, decreased ATP, rapid mitochondrial membrane depolarization, and a rapid activation of calpain but not caspase activity. In the presence of the NMDA receptor antagonist MK-801, 3NP did not induce a significant elevation of [Ca 2+] c within the 24 h time period examined, nor increase [Ca 2+] m within 1 h. ATP was maintained at control levels during the first hour of treatment, but declined 64% by 16 h. Calpain and caspase activity were first evident at 24 h following 3NP administration. 3NP treatment alone resulted in a more rapid decline in ATP, more rapid calpain activation (within 8 h), and elevated [Ca 2+] m as compared to the results obtained with added MK-801. Together, the results demonstrate that 3NP-induced necrotic neuron death is associated with a massive calcium influx through NMDA receptors, resulting in mitochondrial depolarization and calpain activation; while 3NP-induced apoptotic neuron death is not associated with significant elevations in [Ca 2+] c, nor with early changes in [Ca 2+] m, mitochondrial membrane potential, ATP levels, or calpain activity.

Characterization of astrocytes derived from human NTera-2/D1 embryonal carcinoma cells.

Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via high-affinity glutamate transporters, glutamate transporter-1 (GLT-1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate-catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera-2 (NT2/D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4-week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell-cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5-bromo-2'deoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100beta. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT-1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT-1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of (3)H-glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non-toxic, suggesting that these cells were capable of converting it to non-toxic metabolites. NT2/A and NT2-derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.

Cellular Diversity in Mouse Neocortex Revealed by Multispectral Analysis of Amino Acid Immunoreactivity

Cortical cells were classified using an unsupervised cluster analysis based upon their quantitative and combinatorial immunoreactivity for glutamate, gamma-aminobutyric acid (GABA), aspartate, glutamine and taurine. Overall, cell class-specific amino acid signatures were found for 12 cellular types; seven GABA-immunoreactive (GABA-IR) populations (GABA1--7), three classes containing high glutamate levels (GLUT1--3) and two putative glial (GLIA1, 2) cell types. From their large somata, associated vertical processes and high glutamate content, the GLUT classes most probably correspond to pyramidal neurons. Two of the GLUT classes demonstrated complementary distributions in different cortical layers, suggesting spatial separation of cells differing in amino acid immunoreactivity. Of the seven GABA classes, two comprised cells with large somata and displayed medium to low glutamate levels. On the basis of size, these two populations may correspond to large basket cell interneurons. Glial populations could be divided into two classes: GLIA1 cells were more frequently associated with blood vessels and GLIA2 cells were more commonly seen in the lower cortical layers. This work demonstrates that signature recognition based upon amino acid content can be used to separate cortical cells into different categories and reveal further subclasses within these categories. This approach is complementary to other methods using physiological and molecular tools and ultimately will enhance our understanding of neuronal heterogeneity.

Intron 7 retention and exon 9 skipping EAAT2 mRNA variants are not associated with amyotrophic lateral sclerosis

Glutamate-mediated excitotoxicity is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). The astroglial glutamate transporter EAAT2 plays a major role in maintaining low levels of extracellular glutamate in the central nervous system. Multiple EAAT2 mRNA transcripts have been described, but those retaining intron 7 or skipping exon 9 are reported to be specific to the motor cortex, spinal cord, and cerebrospinal fluid of ALS patients. We sought to verify these findings using a TaqMan (Elmer Biosystems, Warrington, UK) real-time reverse transcriptase polymerase chain reaction assay, which provides a sensitive and reliable quantitative measure of EAAT2 transcript copy ratios. We analyzed RNA extracted from frozen postmortem tissue from affected and unaffected central nervous system regions dissected from 17 sporadic ALS patients, 7 Alzheimer's disease patients, and 19 control subjects. We have demonstrated unequivocally that intron 7 retaining and exon 9 skipping variants can be detected in all individuals and in all central nervous system regions studied. The mean ratio of "variant" to "normal" transcripts did not differ significantly between patient and control groups. Although our assay could detect transcript concentrations in cerebrospinal fluid as low as 10 pg/ml, none were detected in 17 ALS and 8 control samples. We conclude that ALS is not associated with elevated levels of EAAT2 transcripts retaining intron 7 and skipping exon 9. An alternative explanation must be sought for the disturbance of glutamate homeostasis reported in ALS.

Glutamate, GABA and precursor amino acids in adult mouse neocortex: cellular diversity revealed by quantitative immunocytochemistry.

Glutamate is an important amino acid in the neocortex for metabolic and neurotransmitter functions. The objective of this study was to detect variations in cellular glutamate content using quantitative immunocytochemistry. We show that glutamate is present in almost all cortical cells and coexists with other amino acids such as aspartate, glutamine or gamma-aminobutyric acid (GABA). The patterns of aspartate and glutamine content suggests that there are no purely aspartatergic or glutaminergic neurons. GABAergic neurons showed variable levels of the precursors such as glutamate, glutamine and aspartate. Comparison of immunoreactive patterns between two cortical areas did not detect any statistically significant differences. The mean cellular intensity for GABA and glutamate was constant across different layers. Surprisingly, we found that GABAergic neurons could coexist with either low or high levels of glutamate, suggesting that metabolic levels of glutamate in these neurons could be variable. Alternatively, some GABA neurons may utilize both GABA and glutamate for neurotransmission. We show that when variations in amino acid content are separately mapped onto individual cells, co-registration is a useful technique for reporting heterogeneity among cortical cells.

Impaired Glutamate Clearance as a Consequence of Energy Failure Caused by MPP+ in Astrocytic Cultures

Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP+) metabolite. The mechanism by which MPP+ is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP+, glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP+ and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP+-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent α-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP+ on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP+ impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.