Evidence for astrocytes as a potential source of the glutamate excess in temporal lobe epilepsy

Increased extracellular brain glutamate has been implicated in the pathophysiology of human refractory temporal lobe epilepsy (TLE), but the cause of the excessive glutamate is unknown. Prior studies by us and others have shown that the glutamate degrading enzyme glutamine synthetase (GS) is deficient in astrocytes in the epileptogenic hippocampal formation in a subset of patients with TLE. We have postulated that the loss of GS in TLE leads to increased glutamate in astrocytes with elevated concentrations of extracellular glutamate and recurrent seizures as the ultimate end-points. Here we test the hypothesis that the deficiency in GS leads to increased glutamate in astrocytes. Rats were chronically infused with methionine sulfoximine (MSO, n=4) into the hippocampal formation to induce GS deficiency and recurrent seizures. A separate group of rats was infused with 0.9% NaCl (saline) as a control (n=6). At least 10days after the start of infusion, once recurrent seizures were established in the MSO-treated rats, the concentration of glutamate was assessed in CA1 of the hippocampal formation by immunogold electron microscopy. The concentration of glutamate was 47% higher in astrocytes in the MSO-treated vs. saline-treated rats (p=0.02), and the ratio of glutamate in astrocytes relative to axon terminals was increased by 74% in the MSO-treated rats (p=0.003). These data support our hypothesis that a deficiency in GS leads to increased glutamate in astrocytes. We additionally propose that the GS-deficient astrocytes in the hippocampal formation in TLE lead to elevated extracellular brain glutamate either through decreased clearance of extracellular glutamate or excessive release of glutamate into the extracellular space from these cells, or a combination of the two.     

Intracellular Na+ concentration influences short-term plasticity of glutamate transporter-mediated currents in neocortical astrocytes

Fast synaptic transmission requires a rapid clearance of the released neurotransmitter from the extracellular space. Glial glutamate transporters (excitatory amino acid transporters, EAATs) strongly contribute to glutamate removal. In this work, we investigated the paired-pulse plasticity of synaptically activated, glutamate transporter-mediated currents (STCs) in cortical layer 2/3 astrocytes. STCs were elicited by local electrical stimulation in layer 4 in the presence of ionotropic glutamate (AMPA and NMDA), GABAA, and GABAB receptor antagonists. In experiments with low [Na(+)]i (5 mM) intrapipette solution, STCs elicited by paired-pulse stimulation demonstrated paired-pulse facilitation (PPF) at short (<250 ms) interstimulus intervals (ISIs) and paired-pulse depression at longer ISIs. In experiments with close to physiological, high [Na(+)]i (20 mM) intrapipette solution, PPF of STCs at short ISIs was significantly reduced. In addition, the STC kinetics was slowed in the presence of high [Na(+)]i. Exogenous GABA increased astrocytic [Na(+)]i, reduced the mean STC amplitude, decreased PPF at short ISIs, and slowed STC kinetics. All GABA-induced changes were blocked by NO-711 and SNAP-5114, GABA transporter (GATs) antagonists. In experiments with the low intrapipette solution, GAT blockade under control conditions decreased PPF at short ISIs both at room and at near physiological temperatures. Dialysis of single astrocyte with low [Na(+)]i solution increased the amplitude and reduced PPR of evoked field potentials recorded in the vicinity of the astrocyte. We conclude that (1) endogenous GABA via GATs may influence EAAT functioning and (2) astrocytic [Na(+)]i modulates the short-term plasticity of STCs and in turn the efficacy of glutamate removal.     

Metabotropic glutamate receptor agonists reduce glutamate release from cultured astrocytes

Astrocytes are thought to control extracellular glutamate concentrations ([Glu]_o) in the brain, thereby protecting neurons from excitotoxic injury. We investigated the effects of metabotropic glutamate receptor (mGluR) agonists on glutamate transport and [Glu]_o in primary hippocampal astrocytic cultures. Acute or chronic exposure of astrocytes to the mGluR agonist trans‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (trans‐ACPD) or its active isomer 1S,3R‐ACPD reduced [Glu]_o in a time‐ and dose‐dependent manner (44.5 ± 3.6% reductions of [Glu]_o in astrocytes from P0–P10 rats and 65.9 ± 4.1 % from rats P20 by 100 μM 1S,3R‐ACPD, EC₅₀ ∼ 5 μM). 1S,3R‐ACPD effects developed slowly (median effective at ∼60 min) and persisted for several hours after agonist removal. ACPD‐pretreated astrocytes established lower steady‐state [Glu]_o levels. ACPD effects persisted in the presence of the glutamate uptake inhibitors D ,L ‐threo‐β‐hydroxyaspartate (THA) and L ‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC) but were impaired by disruption of the transmembrane Na⁺, K⁺, or H⁺ gradients. In addition, 1S,3R‐ACPD had no effects on intracellular glutamate content and did not directly block glutamate transport. Furthermore, ACPD effects could be mimicked by glutamate per se and several other compounds presumed to be mGluR agonists, although (S)‐3,5‐dihydroxyphenylglycine (DHPG), (2S,2R,3R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV), and L ‐(+)‐2‐amino‐4‐phosphonobutyric acid (L ‐AP4) were without effect. These data suggest that glutamate and certain mGluR agonists may regulate [Glu]_o by modulating the transmembrane equilibrium of glutamate transport, especially by attenuating glutamate release. GLIA 25:270–281, 1999. © 1999 Wiley‐Liss, Inc.     

Persistent increase in ventral hippocampal long-term potentiation by juvenile stress: A role for astrocytic glutamine synthetase

A traumatic childhood is among the most important risk factors for developing stress-related psychopathologies such as posttraumatic stress disorder or depression later in life. However, despite the proven role of astrocytes in regulating transmitter release and synaptic plasticity, the contribution of astrocytic transmitter metabolism to such stress-induced psychopathologies is currently not understood. In rodents, childhood adversity can be modeled by juvenile stress exposure, resulting in increased anxiety, and impaired coping with stress in adulthood. We describe that such juvenile stress in rats, regardless of additional stress in adulthood, leads to reduced synaptic efficacy in the ventral CA1 (vCA1) Schaffer collaterals, but increased long-term potentiation (LTP) of synaptic transmission after high-frequency stimulation. We tested whether the glutamate–glutamine-cycle guides the lasting changes on plasticity observed after juvenile stress by blocking the astrocytic glutamate-degrading enzyme, glutamine synthetase (GS). Indeed, the pharmacological inhibition of GS by methionine sulfoximine in slices from naïve rats mimics the effect of juvenile stress on vCA1-LTP, while supplying glutamine is sufficient to normalize the LTP. Assessing steady-state mRNA levels in the vCA1 stratum radiatum reveals distinct shifts in the expression of GS, astrocytic glutamate, and glutamine transporters after stress in juvenility, adulthood, or combined juvenile/adult stress. While GS mRNA expression levels are lastingly reduced after juvenile stress, GS protein levels are maintained stable. Together our results suggest a critical role for astrocytes and the glutamate–glutamine cycle in mediating long-term effects of juvenile stress on plasticity in the vCA1, a region associated with anxiety and emotional memory processing.     

Dual regulation of Ca2+‐dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation‐induced Ca²⁺‐dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca²⁺‐dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca²⁺ increase. Inhibition of glutamine synthetase activity by L ‐methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca²⁺‐dependent glutamate release, without affecting intracellular Ca²⁺ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca²⁺‐dependent release of glutamate from astrocytes. © 2009 Wiley‐Liss, Inc.     

Mechanisms regulating spill‐over of synaptic glutamate to extrasynaptic NMDA receptors in mouse substantia nigra dopaminergic neurons

N‐Methyl‐d ‐aspartate glutamate receptors (NMDARs) contribute to neural development, plasticity and survival, but they are also linked with neurodegeneration. NMDARs at synapses are activated by coincident glutamate release and depolarization. NMDARs distal to synapses can sometimes be recruited by ‘spill‐over’ of glutamate during high‐frequency synaptic stimulation or when glutamate uptake is compromised, and this influences the shape of NMDAR‐mediated postsynaptic responses. In substantia nigra dopamine neurons, activation of NMDARs beyond the synapse during different frequencies of presynaptic stimulation has not been explored, even though excitatory afferents from the subthalamic nucleus show a range of firing frequencies, and these frequencies change in human and experimental Parkinson's disease. This study reports that high‐frequency stimulation (80 Hz/200 ms) evoked NMDAR‐excitatory postsynaptic currents (EPSCs) that were larger and longer lasting than those evoked by single stimuli at low frequency (0.1 Hz). MK‐801, which irreversibly blocked NMDAR‐EPSCs activated during 0.1‐Hz stimulation, left a proportion of NMDAR‐EPSCs that could be activated by 80‐Hz stimulation and that may represent activity of NMDARs distal to synapses. TBOA, which blocks glutamate transporters, significantly increased NMDAR‐EPSCs in response to 80‐Hz stimulation, particularly when metabotropic glutamate receptors (mGluRs) were also blocked, indicating that recruitment of NMDARs distal to synapses is regulated by glutamate transporters and mGluRs. These regulatory mechanisms may be essential in the substantia nigra for restricting glutamate diffusion from synaptic sites and keeping NMDAR‐EPSCs in dopamine neurons relatively small and fast. Failure of glutamate transporters may contribute to the declining health of dopamine neurons during pathological conditions.     

Astrocytic pathology of methionine sulfoximine-induced encephalopathy

Summary To investigate the roles imposed on astrocytes for glutamate metabolism, a specific inhibitor of glutamine synthetase (GS), methionine sulfoximine (MSO), was repeatedly administered to rats and histopathological changes were correlated with glycogen accumulation and the immunocytochemistry of GS and glial fibrillary acidic protein (GFAP). Prolonged MSO-loading (every 12 h up to three times, 100–150 mg/kg body weight) brought about the appearance of astrocytes with swollen, watery nuclei reminiscent of Alzheimer II glia chiefly in the neocortex, hippocampus and lateral thalamus after 24 h. Concomitantly, profound accumulation of glycogen ensued in the superficial three layers of the neocortex, hippocampus and pyriform cortex. GS immunoreactivity appeared enhanced in the cortex, hippocampus and lateral thalamus with parallel increase in GFAP immunoreactivity after prolonged treatment. Oligodendrocytes in the diencephalon and brain stem also normally contained GS immunoreactivity. Some animals developed necrotic lesions in the dorsolateral neocortex. The area of glycogen accumulation coincided with the known distribution ofN-methyld-aspartate (NMDA) glutamate receptors and, thus, GS may play important roles in NMDA receptor-mediated glutamate metabolism. The Alzheimer II type changes, however, did not correlate with NMDA-receptor distribution. These results indicate certain regionalizations in the roles of astrocytes and oligodendrocytes in glutamate and ammonia metabolisms.Supported by the grants-in-aid from the Ministry of Education, Science and Culture (Projects 63480214 and 63870063 to T. Yamamoto), and from the Ministry of Health (for Neurodegenerative disorders to T. Yamamoto), Japan     

Sex differences in the glutamate signaling pathway in juvenile rats

Females have been found to be at lower risk for the development of neurodevelopmental disorders than males. The greater neuroprotection in females is mostly due to female sex hormones. Estrogen is hypothesized to provide neuroprotection by suppressing the neuro‐excitotoxicity induced by glutamate (Glu). This study was conducted to understand the effect of sex in modulating Glu signaling in juvenile rats. Brain tissue homogenate of 15 Wistar albino rats (9 males, 6 females) weighing 60 to 80 g and aged approximately 28 days was used. Biochemical parameters related to Glu signaling, such as the absolute and relative concentrations of Glu, gamma aminobutyric acid (GABA), and glutamine, as well as glutamate transporter 1 (GLT1), glutamine synthetase (GS), glutaminase (GLN), and glutamate decarboxylase‐67 (GAD‐67), were measured by ELISA. The data obtained demonstrated that compared with the levels in males, female rats exhibited significantly lower levels of Glu (p = .001) and GLN/GS (p = .021). The Glu/GABA and Glu/GLT1 ratios as well as the levels of GAD‐67 were also lower in female rats, although the difference was not significant. The GLN/GAD‐67 ratio (p = .027) and levels of GS (p = .019) were significantly higher in female rats than in males. Multiple regression analysis confirmed the role of GLN/GS, together with the much higher affinity of GLT1 to Glu, in avoiding excitotoxicity in females. In conclusion, there was a significant difference in Glu signaling between female and male rats. The females exhibited a lower susceptibility to develop Glu‐induced excitotoxicity, an etiological mechanism for multiple neurodevelopmental disorders.     

The effect of methamphetamine on the release of glutamate from striatal slices

Summary One postulated role of dopamine in the striatum is to reduce neuronal activity in cortico-striatal glutamergic terminals. We investigated the effects of methamphetamine, which displaces dopamine, on glutamate release from rat striatal slices. Methamphetamine significantly reduced K⁺-stimulated (45 mM) glutamate release. In slices prepared from rats treated 8 days previously with methamphetamine there was enhanced (approximately 200%) release of glutamate. This study demonstrates that dopamine has a modulatory effect on glutamate release in the striatum.     

Brain alanine formation as an ammonia-scavenging pathway during hyperammonemia: effects of glutamine synthetase inhibition in rats and astrocyte–neuron co-cultures

Hyperammonemia is a major etiological toxic factor in the development of hepatic encephalopathy. Brain ammonia detoxification occurs primarily in astrocytes by glutamine synthetase (GS), and it has been proposed that elevated glutamine levels during hyperammonemia lead to astrocyte swelling and cerebral edema. However, ammonia may also be detoxified by the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT) leading to trapping of ammonia in alanine, which in vivo likely leaves the brain. Our aim was to investigate whether the GS inhibitor methionine sulfoximine (MSO) enhances incorporation of ¹⁵NH₄⁺ in alanine during acute hyperammonemia. We observed a fourfold increased amount of ¹⁵NH₄ incorporation in brain alanine in rats treated with MSO. Furthermore, co-cultures of neurons and astrocytes exposed to ¹⁵NH₄Cl in the absence or presence of MSO demonstrated a dose-dependent incorporation of ¹⁵NH₄ into alanine together with increased ¹⁵N incorporation in glutamate. These findings provide evidence that ammonia is detoxified by the concerted action of GDH and ALAT both in vivo and in vitro, a mechanism that is accelerated in the presence of MSO thereby reducing the glutamine level in brain. Thus, GS could be a potential drug target in the treatment of hyperammonemia in patients with hepatic encephalopathy.     

Transporter‐mediated replacement of extracellular glutamate for GABA in the developing murine neocortex

During early development, cortical neurons migrate from their places of origin to their final destinations where they differentiate and establish synaptic connections. During corticogenesis, radially migrating cells move from deeper zone to the marginal zone, but they do not invade the latter. This “stop” function of the marginal zone is mediated by a number of factors, including glutamate and γ‐aminobutyric acid (GABA), two main neurotransmitters in the central nervous system. In the marginal zone, GABA has been shown to be released via GABA transporters (GAT)‐2/3, whereas glutamate transporters (EAATs) operate in the uptake mode. In this study, GABAergic postsynaptic currents (GPSCs) were recorded from Cajal‐Retzius cells in the marginal zone of murine neonatal neocortex using a whole‐cell patch‐clamp technique. Minimal electrical stimulation was applied to elicit evoked GPSCs using a paired‐pulse protocol. EAAT blockade with dl ‐threo‐b‐benzyloxyaspartic acid (dl ‐TBOA), a specific non‐transportable EAAT antagonist, abolishes constitutive GAT‐2/3‐mediated GABA release. In contrast to dl ‐TBOA, d ‐aspartate, an EAAT substrate, fails to block GAT‐2/3‐mediated GABA release. SNAP‐5114, a specific GAT‐2/3 antagonist, induced an elevation of intracellular sodium concentration ([Na⁺]_i) under resting conditions and in the presence of d ‐aspartate, indicating that GAT‐2/3 operates in reverse mode. In the presence of dl ‐TBOA, however, SNAP‐5114 elicited a [Na⁺]_i decrease, demonstrating that GAT‐2/3 operates in uptake mode. We conclude that EAATs via intracellular Na⁺ signaling and/or cell depolarization can govern the strength/direction of GAT‐mediated GABA transport.     

Toward feedback controlled deep brain stimulation: Dynamics of glutamate release in the subthalamic nucleus in rats

Deep brain stimulation (DBS) is an effective symptomatic treatment in Parkinson's disease. High frequency stimulation (HFS) of the subthalamic nucleus elicits neurotransmitter release in multiple nuclei. Therefore, we tested the hypothesis that neurotransmitter release during HFS may be used to provide feedback control of the intensity and pattern of HFS. We studied the dynamic relationship between extracellular glutamate levels and HFS in and around the STN in anesthetized rats. We used a pseudorandom binary sequence (PRBS) of stimulation in the STN, the independent forcing function, while measuring extracellular glutamate in the same nucleus, the dependent variable. The PRBS consisted of 90 s periods during which stimulation (100 μA, 150 Hz, 10% duty cycle) was either off or on. The stimulation and extracellular glutamate levels were fitted using an autoregressive exogenous model (ARX) to determine the transfer function between HFS and the extracellular glutamate concentration in the STN. The ARX model fit the dynamics of extracellular glutamate levels well (correlation coefficients ranged from 0.74 to 0.99; n = 11). The transfer function accurately predicted extracellular glutamate levels in the STN even when the pattern of HFS was modified. We used the transfer function to develop a feedback controlled stimulation algorithm. Feedback controlled HFS maintained extracellular glutamate concentrations at any predefined level, but only intermittent HFS was required. We conclude that the transfer function between HFS and neurotransmitter levels in the brain can be used to design DBS protocols that generate specific temporal patterns of glutamate release in the STN.     

Altered expression of glutamate transporters under hypoxic conditions in vitro

Regulation of extracellular excitotoxins by glial and neuronal glutamate transporters is critical to maintain synaptic terminal integrity. Factors interfering with the normal functioning of these transporters might be involved in neurodegeneration. Among them, recent studies have shown that hypoxia alters glutamate transporter function; however, it is unclear if hypoxia has an effect on the expression of glutamate transporters and which intracellular signaling pathways are involved. The C6 rat glial and GT1--7 mouse neuronal cell lines were exposed to hypoxic conditions (5% CO(2), 95% N(2)) and levels of glutamate transporter mRNA were determined by ribonuclease protection assay. After 21 hr, there was a 100% increase in levels of rat excitatory amino acid transporter 3 (EAAT3) mRNA in C6 cells and a 600% increase in levels of murine EAAT2 mRNA in GT1--7 cells. There was a similar increase in mRNA levels after hypoxia in C6 cells transfected with human EAAT2, whereas reoxygenation normalized the expression levels of glutamate transporters. Although the expression of EAATs was associated with increased immunoreactivity by Western blot, functioning of the transporters was decreased as evidenced by D-aspartate uptake. Finally, although the protein kinase C stimulator phorbol-12-myristate-13-acetate enhanced EAAT2 mRNA levels after hypoxia, protein kinase C inhibitor bisindolylmaleimide I had the opposite effect. Taken together, this study suggests that the hypoxia is capable of upregulating levels of EAATs via a protein kinase C-dependent compensatory mechanism. This increased expression is not sufficient to overcome the decreased functioning of the EAATs associated with decreased ATP production and mitochondrial dysfunction.     

Cystine/glutamate antiporter blockage induces myelin degeneration

The cystine/glutamate antiporter is a membrane transport system responsible for the uptake of extracellular cystine and release of intracellular glutamate. It is the major source of cystine in most cells, and a key regulator of extrasynaptic glutamate in the CNS. Because cystine is the limiting factor in the biosynthesis of glutathione, and glutamate is the most abundant neurotransmitter, the cystine/glutamate antiporter is a central player both in antioxidant defense and glutamatergic signaling, two events critical to brain function. However, distribution of cystine/glutamate antiporter in CNS has not been well characterized. Here, we analyzed expression of the catalytic subunit of the cystine/glutamate antiporter, xCT, by immunohistochemistry in histological sections of the forebrain and spinal cord. We detected labeling in neurons, oligodendrocytes, microglia, and oligodendrocyte precursor cells, but not in GFAP⁺ astrocytes. In addition, we examined xCT expression and function by qPCR and cystine uptake in primary rat cultures of CNS, detecting higher levels of antiporter expression in neurons and oligodendrocytes. Chronic inhibition of cystine/glutamate antiporter caused high toxicity to cultured oligodendrocytes. In accordance, chronic blockage of cystine/glutamate antiporter as well as glutathione depletion caused myelin disruption in organotypic cerebellar slices. Finally, mice chronically treated with sulfasalazine, a cystine/glutamate antiporter inhibitor, showed a reduction in the levels of myelin and an increase in the myelinated fiber g‐ratio. Together, these results reveal that cystine/glutamate antiporter is expressed in oligodendrocytes, where it is a key factor to the maintenance of cell homeostasis. GLIA 2016. GLIA 2016;64:1381–1395     

Glutamine synthetase becomes nitrated and its activity is reduced during repetitive seizure activity in the pentylentetrazole model of epilepsy

Purpose: The astrocyte‐specific glutamine synthetase (GS) plays a key role in glutamate recycling and Gamma‐aminobutyric acid (GABA) metabolism. Changes in the expression or activity of GS have been proposed to contribute to epileptogenesis. The mechanisms or how and where GS may contribute to epilepsy is still a matter of discussion. Here we asked the question whether brain regions, which show an astrocytic stress response respond with alterations of GS. Methods: Biochemical and histological alterations of GS, HSP‐27, and GFAP were studied after pentylenetetrazole‐induced repetitive epileptic seizures (PIRS) in rats using a topographical quantification of the GS‐immunoreactivity (GSIR) in relation to the focal heat shock response (HSR). Saline‐treated rats served as controls and rats treated by the GS‐inhibitor, L‐methionine‐sulfoximine (MSO) served as a positive control. Results: No changes in the amount of GSIR and GS‐protein occurred during PIRS. A significant reduction of GSIR was observed by histochemistry (in situ) and in native (nonheated) protein extracts of MSO‐treated rats. In rats affected by PIRS, GS‐activity showed a significant, region‐specific reduction in association with a nitration of the enzyme. Discussion: These results show that neither PIRS nor GS‐inhibition reduced the amount of GS protein, but that MSO interferes with antibody binding to native GS. PIRS resulted in a focal increase of astrocytic stress response, whereas MSO caused a widespread, homogeneous astrocytic HSR independent from quantitative changes of GS content. In rats with PIRS the regions showing a strong glial HSR, respond with reduced GS‐activity and GS‐nitration, which all together are clear indicators of a nitrosative stress response.     

Deficit in hippocampal long-term potentiation in monosodium glutamate-treated rats

Rats subjected to monosodium glutamate (MSG) administration during the neonatal period present chronic neuroendocrine dysfunction associated with marked cognitive deficits. Long-term potentiation (LTP) in the hippocampus provides a model suited for the study of mammalian brain plasticity and memory formation. In the present work, we used the LTP protocol to investigate the synaptic plasticity in the hippocampal CA1 area of adult rats subjected to MSG treatment during the first 10 days of life. Synaptic transmission in CA1 area was analyzed using extracellular field recordings in response to Schaffer's collateral fiber stimulation in hippocampal slices. Animals injected with MSG exhibited a dramatic decrement of LTP field excitatory postsynaptic potentials (fEPSPs) compared to control group. Analysis of percent enhancement of fEPSP slope at 2 min after high frequency stimulation (HFS) increased by 189.3 +/- 33.2% in slices from control rats and 129.45 +/- 18.5% (p < 0.01) in slices from MSG-treated rats. Additionally, MSG-treated animals failed to maintain or consolidate LTP as revealed by a significant reduction in fEPSP slope enhancement over time after HFS. The mean fEPSP slope, 60 min after HFS, was 154.28 +/- 21% of the average baseline slope in control slices versus only 124.4 +/- 15% in MSG-treated rats (p < 0.01). At 90 min after HFS, slices from controls reached a potentiation of 44.5 +/- 2.9%, whereas the MSG group displayed an overall response enhancement of 17.65 +/- 2.7% of basal levels (p < 0.01). These findings indicate that MSG-treated rats display a chronic impairment of CA1 synaptic plasticity.     

Differential mechanisms of Ca2+ responses in glial cells evoked by exogenous and endogenous glutamate in rat hippocampus

The mechanisms of Ca2+ responses evoked in hippocampal glial cells in situ, by local application of glutamate and by synaptic activation, were studied in slices from juvenile rats using the membrane permeant fluorescent Ca2+ indicator fluo-3AM and confocal microscopy. Ca2+ responses induced by local application of glutamate were unaffected by the sodium channel blocker tetrodotoxin and were therefore due to direct actions on glial cells. Glutamate-evoked responses were significantly reduced by the L-type Ca2+ channel blocker nimodipine, the group I/II metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)2-amino-5-phosphonopentanoic acid (APV). However, glutamate-induced Ca2+ responses were not significantly reduced by the non-NMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). These results indicate that local application of glutamate increases intracellular Ca2+ levels in glial cells via the activation of L-type Ca2+ channels, NMDA receptors, and metabotropic glutamate receptors. Brief (1 s) tetanization of Schaffer collaterals produced increases in intracellular Ca2+ levels in glial cells that were dependent on the frequency of stimulation (> or =50 Hz) and on synaptic transmission (abolished by tetrodotoxin). These Ca2+ responses were also antagonized by the L-type Ca2+ channel blocker nimodipine and the metabotropic glutamate receptor antagonist MCPG. However, the non-NMDA receptor antagonist CNQX significantly reduced the Schaffer collateral-evoked Ca2+ responses, while the NMDA antagonist APV did not. Thus, these synaptically mediated Ca2+ responses in glial cells involve the activation of L-type Ca2+ channels, group I/II metabotropic glutamate receptors, and non-NMDA receptors. These findings indicate that increases in intracellular Ca2+ levels induced in glial cells by local glutamate application and by synaptic activity share similar mechanisms (activation of L-type Ca2+ channels and group I/II metabotropic glutamate receptors) but also have distinct components (NMDA vs. non-NMDA receptor activation, respectively). Therefore, neuron-glia interactions in rat hippocampus in situ involve multiple, complex Ca2+-mediated processes that may not be mimicked by local glutamate application.     

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non‐cell‐autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1^G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1^G93A rats, we have demonstrated that the typical Ca²⁺ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca²⁺ concentration. Since the expression of protein kinase C epsilon isoform (PKC?) has been found to be considerably reduced in astrocytes from hSOD1^G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKC? expression was found to restore Ca²⁺ oscillations induced by mGluR5 activation in hSOD1^G93A‐expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKC? expression in astrocytes from wild‐type animals with specific PKC?‐shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake‐mediated by mGluR5 activation. These results suggest that PKC? is required to generate Ca²⁺ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKC? could impair this neuroprotective process and participate in the progression of ALS.     

Over-expression of the human EAAT2 glutamate transporter within neurons of mouse organotypic hippocampal slice cultures leads to increased vulnerability of CA1 pyramidal cells

Excitatory amino acid transporters (EAATs) maintain the balance between pathological and physiological conditions by limiting the extracellular concentration of glutamate within the CNS and thus preventing excitotoxic injury. The loss of EAAT2 has been associated with the development of neurological diseases such as amyotrophic lateral sclerosis. It has therefore been suggested that the over-expression of specific EAATs may provide some degree of neuroprotection. However, the inability to isolate and study the function of the different EAAT isoforms in a cell type-specific manner has made it difficult to determine the exact contribution of individual EAATs toward neuroprotection or neurodegeneration in the context of excitotoxic injury. To address this question, we transduced hippocampal slice cultures from 1-week-old C57B/6 mice with recombinant adeno-associated virus carrying an EAAT2 gene expression cassette. EAAT2 gene expression was driven in neurons with the neuron-specific enolase promoter. Using this model system, we were able to induce a significant increase in the expression of functional EAAT2. Consequently, a significant increase in CA1 neuronal damage was observed in slices over-expressing EAAT2 in neurons following an acute exposure to exogenous glutamate. These data suggest that the increased expression of EAAT2 within neurons may contribute to neurodegeneration.     

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full‐field light of various intensities. Light‐evoked membrane potential changes (ΔV_m) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark‐adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage‐gated Ca²⁺ influx (Q^Ca) and consequent glutamate release. Stimulus‐evoked glutamate release was quantified by the increase in membrane capacitance (ΔC_m). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔV_m/Q^Ca ratio equally at a given light intensity and inhibition did not alter the overall relation between Q^Ca and ΔC_m. However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔC_m unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non‐linear relationship between Q^Ca and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.