In vivo neuroprotective role of NMDA receptors against kainate-induced excitotoxicity in murine hippocampal pyramidal neurons

Activation of NMDA receptors has been shown to induce either neuronal cell death or neuroprotection against excitotoxicity in cultured cerebellar granule neurons in vitro. We have investigated the effects of pretreatment with NMDA on kainate-induced neuronal cell death in mouse hippocampus in vivo. The systemic administration of kainate (30 mg/kg), but not NMDA (100 mg/kg), induced severe damage in pyramidal neurons of the hippocampal CA1 and CA3 subfields 3-7 days later, without affecting granule neurons in the dentate gyrus. An immunohistochemical study using an anti-single-stranded DNA antibody and TdT-mediated dUTP nick end labeling analysis both revealed that kainate, but not NMDA, induced DNA fragmentation in the CA1 and CA3 pyramidal neurons 1-3 days after administration. Kainate-induced neuronal loss was completely prevented by the systemic administration of NMDA (100 mg/kg) 1 h to 1 day previously. No pyramidal neuron was seen with fragmented DNA in the hippocampus of animals injected with kainate 1 day after NMDA treatment. The neuroprotection mediated by NMDA was prevented by the non-competitive NMDA receptor antagonist MK-801. Taken together these results indicate that in vivo activation of NMDA receptors is capable of protecting against kainate-induced neuronal damage through blockade of DNA fragmentation in murine hippocampus.     

Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons

Glutamate neurotoxicity is thought to play a role in the pathogenesis of several neurodegenerative diseases. While prolonged activation of either NMDA or non-NMDA receptors causes neuronal damage, NMDA receptors appear to mediate most of the glutamate toxicity. The reasons why NMDA toxicity predominates are uncertain but may relate to more effective neuroprotective mechanisms acting at non-NMDA receptors. To determine whether desensitization is one such mechanism, we studied the effects of the lectin wheat germ agglutinin (WGA) on quisqualate currents and toxicity in cultured postnatal rat hippocampal neurons. After WGA treatment, quisqualate currents exhibit little desensitization and a 4- to 8-fold increase in steady-state amplitude. WGA also markedly augments the degree of acute, quisqualate-induced neuronal degeneration. These results suggest that non-NMDA desensitization serves a neuroprotective function in hippocampal neurons.     

Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation.

BACKGROUND: Paraneoplastic syndromes are "remote" complications of cancer characterized clinically by neurological disease. The sera and cerebrospinal fluid (CSF) from patients with paraneoplastic neurological syndromes (PNS) frequently contain autoantibodies to ill-defined neuronal antigens. We report here that neuronal glutamate receptors are targets for autoantibodies found in the serum from some patients with well-characterized PNS. MATERIALS AND METHODS: We have analyzed the serum from seven patients with well-characterized PNS for the presence of autoreactive antibodies to non-NMDA glutamate receptor subunits. Autoantibodies were assessed using Western blot, immunohistochemistry, and immunocytochemistry. Whole-cell electrophysiological recordings were used to examine the effect of antibodies on glutamate receptors expressed by cortical neurons in culture. RESULTS: Six of seven patients' serum contained autoantibodies to the non-NMDA glutamate receptor (GluR) subunits GluR1, GluR4, and/or GluR5/6. No patient had autoantibodies to GluR2, and only one patient exhibited weak immunoreactivity to GluR3. Electrophysiological analysis demonstrated that the serum from four of the six GluR-antibody-positive patients enhanced glutamate-elicited currents on cultured cortical neurons but had no effect on receptor function alone. Enhancement of glutamate-elicited currents was also produced by affinity-purified antibody to GluR5. CONCLUSIONS: The occurrence of autoantibodies to specific neuronal neurotransmitter subunits in the sera of patients with PNS and the ability of these autoantibodies to modulate glutaminergic receptor function suggest that some paraneoplastic neurological injury could result from glutamate-mediated excitotoxicity.     

Potassium channel gene therapy can prevent neuron death resulting from necrotic and apoptotic insults

Necrotic insults such as seizure are excitotoxic. Logically, membrane hyperpolarization by increasing outwardly conducting potassium channel currents should attenuate hyperexcitation and enhance neuron survival. Therefore, we overexpressed a small-conductance calcium-activated (SK2) or voltage-gated (Kv1.1) channel via viral vectors in cultured hippocampal neurons. We found that SK2 or Kv1.1 protected not only against kainate or glutamate excitotoxicity but also increased survival after sodium cyanide or staurosporine. In vivo overexpression of either channel in dentate gyrus reduced kainate-induced CA3 lesions. In hippocampal slices, the kainate-induced increase in granule cell excitability was reduced by overexpression of either channel, suggesting that these channels exert their protective effects during hyperexcitation. It is also important to understand any functional disturbances created by transgene overexpression alone. In the absence of insult, overexpression of Kv1.1, but not SK2, reduced baseline excitability in dentate gyrus granule cells. Furthermore, while no behavioral disturbances during spatial acquisition in the Morris water maze were observed with overexpression of either channel, animals overexpressing SK2, but not Kv1.1, exhibited a memory deficit post-training. This difference raises the possibility that the means by which these channel subtypes protect may differ. With further development, potassium channel vectors may be an effective pre-emptive strategy against necrotic insults.     

Terfenadine induces toxicity in cultured cerebellar neurons: A role for glutamate receptors

Summary Exposure of cultured cerebellar neurons to the histamine H1 receptor antagonist terfenadine resulted in neuronal degeneration and death. Terfenadine neurotoxicity was dependent upon concentration and time of exposure. After 2h exposure, 20µM terfenadine reduced the number of surviving neurons by 75%, and as low as 10nM terfenadine induced significant neurotoxicity after 5 days of exposure. Neuronal sensitivity to terfenadine changed with age in culture, and at 25 days in culture neurons appeared to be much less sensitive than at 5 or 9–17 days in culture. Neurotoxicity by terfenadine could not be prevented by high concentrations of histamine (5 mM), but it was significantly delayed by blocking NMDA or non-NMDA glutamate receptors with MK-801 or CNQX respectively, suggesting the involvement of excitatory transmission mediated by glutamate in the neurotoxicity induced by terfenadine in these neurons. We also found that the presence of terfenadine (5,µM) unveiled the potential excitotoxicity of the non-NMDA receptor agonist AMPA (100µM), and reduced the concentration of glutamate necessary to induce excitotoxicity, compared to untreated cultures. These results suggest a role for terfenadine in the modulation of the excitotoxic response mediated in cerebellar neurons through ionotropic glutamate receptors.     

Characterization of glutamate toxicity in cultured rat cerebellar granule neurons at reduced temperature

We have defined conditions whereby glutamate becomes toxic to isolated cerebellar granule neurons in a physiologic salt solution (pH 7.4). In the presence of a physiologic Mg⁺⁺ concentration, acute glutamate excitotoxicity manifests only when the temperature was reduced from 37°C to 22°C. In contrast to glutamate, N-methyl-D-aspartate (NMDA) was non-toxic at either temperature at concentrations as high as 1 mM. Glycine strongly potentiated both the potency and efficacy of glutamate but revealed only a modest NMDA response. The non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxalinedione (CNQX), potently protected against glutamate challenge, although the contribution of antagonism at strychnine-insensitive glycine sites could not be excluded. To further characterize the non-NMDA receptor contribution to the excitotoxic response, the promiscuity of glutamate interaction with ionotropic receptors was simulated by exposing neurons to NMDA in the presence of non-NMDA receptor agonists. NMDA toxicity was potentiated four- to sevenfold when non-NMDA receptors were coactivated by a subtoxic concentration of AMPA, kainate, or domoate. These results suggest that non-NMDA receptor activation participates in the mechanism of acute glutamate toxicity by producing neuronal depolarization (via sodium influx), which in turn promotes the release of the voltage-dependent magnesium blockade of NMDA receptor ion channels. © 1997 John Wiley & Sons, Inc.     

Cyclic AMP-Elevating Agents Prevent Oligodendroglial Excitotoxicity

Abstract: Previously, we have demonstrated that cells of the oligodendroglial lineage express non-NMDA glutamate receptor genes and are damaged by kainate-induced Ca²⁺ influx via non-NMDA glutamate receptor channels, representing oligodendroglial excitotoxicity. We find in the present study that agents that elevate intracellular cyclic AMP prevent oligodendroglial excitotoxicity. After oligodendrocyte-like cells, differentiated from the CG-4 cell line established from rat oligodendrocyte type-2 astrocyte progenitor cells, were exposed to 2 mM kainate for 24 h, cell death was evaluated by measuring activity of lactate dehydrogenase released into the culture medium. Released lactate dehydrogenase increased about threefold when exposed to 2 mM kainate. Kainate-induced cell death was prevented by one of the following agents: adenylate cyclase activator (forskolin), cyclic AMP analogues (dibutyryl cyclic AMP and 8-bromo-cyclic AMP), and cyclic AMP phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, pentoxifylline, propentofylline, and ibudilast). Simultaneous addition of both forskolin and phosphodiesterase inhibitors prevented the kainate-induced cell death in an additive manner. A remarkable increase in Ca²⁺ influx (～5.5-fold) also was induced by kainate. The cyclic AMP-elevating agents caused a partial suppression of the kainate-induced increase in Ca²⁺ influx, leading to a less prominent response of intracellular Ca²⁺ concentration to kainate. The suppressing effect of forskolin on the kainate-induced Ca²⁺ influx was partially reversed by H-89, an inhibitor of cyclic AMP-dependent protein kinase. In contrast to this, okadaic acid, an inhibitor of protein phosphatases 1 and 2A, brought about a decrease in the kainate-induced Ca²⁺ influx. We therefore concluded that cyclic AMP-elevating agents prevented oligodendroglial excitotoxicity by cyclic AMP-dependent protein kinase-dependent protein phosphorylation, resulting in decreased kainate-induced Ca²⁺ influx.     

Fast inhibition of glutamate-activated currents by caffeine

Background

Caffeine stimulates calcium-induced calcium release (CICR) in many cell types. In neurons, caffeine stimulates CICR presynaptically and thus modulates neurotransmitter release.

Methodology/Principal Findings

Using the whole-cell patch-clamp technique we found that caffeine (20 mM) reversibly increased the frequency and decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) in neocortical neurons. The increase in mEPSC frequency is consistent with a presynaptic mechanism. Caffeine also reduced exogenously applied glutamate-activated currents, confirming a separate postsynaptic action. This inhibition developed in tens of milliseconds, consistent with block of channel currents. Caffeine (20 mM) did not reduce currents activated by exogenous NMDA, indicating that caffeine block is specific to non-NMDA type glutamate receptors.

Conclusions/Significance

Caffeine-induced inhibition of mEPSC amplitude occurs through postsynaptic block of non-NMDA type ionotropic glutamate receptors. Caffeine thus has both pre and postsynaptic sites of action at excitatory synapses.     

Lithium protection against glutamate excitotoxicity in rat cerebral cortical neurons: involvement of NMDA receptor inhibition possibly by decreasing NR2B tyrosine phosphorylation

The therapeutic mechanisms of lithium for treating bipolar mood disorder remain poorly understood. Recent studies demonstrate that lithium has neuroprotective actions against a variety of insults. Here, we studied neuroprotective effects of lithium against excitotoxicity in cultured cerebral cortical neurons. Glutamate-induced excitotoxicity in cortical neurons was exclusively mediated by NMDA receptors. Pre-treatment of cortical neurons with LiCl time-dependently suppressed excitotoxicity with maximal protection after 6 days of pre-treatment. Significant protection was observed at the therapeutic and subtherapeutic concentration of 0.2-1.6 mm LiCl with almost complete protection at 1 mM. Neuroprotection was also elicited by valproate, another major mood-stabilizer. The neuroprotective effects of lithium coincided with inhibition of NMDA receptor-mediated calcium influx. Lithium pre-treatment did not alter total protein levels of NR1, NR2A and NR2B subunits of NMDA receptors. However, it did markedly reduce the level of NR2B phosphorylation at Tyr1472 and this was temporally associated with its neuroprotective effect. Because NR2B tyrosine phosphorylation has been positively correlated with NMDA receptor-mediated synaptic activity and excitotoxicity, the suppression of NR2B phosphorylation by lithium is likely to result in the inactivation of NMDA receptors and contributes to neuroprotection against excitotoxicity. This action could also be relevant to its clinical efficacy for bipolar patients.     

NMDA and non-NMDA receptor-mediated differential Ca2+ load and greater vulnerability of motor neurons in spinal cord cultures

Glutamate receptor activated neuronal cell death has been implicated in the pathogenesis of motor neuron disease but the molecular mechanism responsible for neuronal dysfunction needs to be elucidated. In the present study, we examined the contribution of NMDA and non-NMDA sub-types of glutamate receptors in selective vulnerability of motor neurons. Glutamate receptor activated Ca2+ signaling, mitochondrial functions and neurotoxicity in motor neurons and other spinal neurons were studied in mixed spinal cord primary cultures. Exposure of cells to glutamate receptor agonists glutamate, NMDA and AMPA elevated the intracellular Ca2+, mitochondrial Ca2+ and caused mitochondrial depolarization and cytotoxicity in both motor neurons and other spinal neurons but a striking difference was observed in the magnitude and temporal patterns of the [Ca2+]i responses between the two neuronal cell types. The motor neurons elicited higher Ca2+ load than the other spinal neurons and the [Ca2+]i levels were elevated for a longer duration in motor neurons. AMPA receptor stimulation was more effective than NMDA. Both the NMDA and non-NMDA receptor antagonists APV and NBQX inhibited the Ca2+ entry and decreased the cell death significantly; however, NBQX was more potent than APV. Our results demonstrate that both NMDA and non-NMDA sub-types of glutamate receptors contribute to glutamate-mediated motor neuron damage but AMPA receptors play the major role. AMPA receptor-mediated excessive Ca2+ load and differential handling/regulation of Ca2+ buffering by mitochondria in motor neurons could be central in their selective vulnerability to excitotoxicity.     

Increased tyrosine phosphorylation of PSD-95 by Src family kinases after brain ischaemia

PSD (postsynaptic density)-95, a scaffold protein that tethers NMDA (N-methyl-D-aspartate) receptors to signal molecules, is implicated in pathological events resulting from excitotoxicity. The present study demonstrates that brain ischaemia and reperfusion increase the tyrosine phosphorylation of PSD-95 in the rat hippocampus. PP2, a specific inhibitor of SrcPTKs (Src family protein tyrosine kinases), prevents the ischaemia-induced increases not only in the tyrosine phosphorylation of PSD-95, but also in the interaction between PSD-95 and Src kinases. PSD-95 is phosphorylated either by purified Src/Fyn kinases in vitro or by co-expression of constitutively active Src/Fyn in COS7 cells. The results suggest that SrcPTKs are involved in PSD-95 phosphorylation. The single Tyr(523) mutation to phenylalanine (Y523F) reduces the Src/Fyn-mediated phosphorylation of PSD-95 in COS7 cells and in vitro. As shown with a rabbit polyclonal antibody against phospho-PSD-95 (Tyr(523)), Tyr(523) phosphorylation is responsible for the increased tyrosine phosphorylation of PSD-95 induced by ischaemia in the rat hippocampus. In cultured hippocampal neurons, overexpression of PSD-95 Y523F, but not PSD-95 Y533F, abolishes the facilitating effect of PSD-95 on the glutamate- or NMDA-mediated currents, implying that PSD-95 Tyr(523) phosphorylation contributes to the post-ischaemic over-activation of NMDA receptors. Thus the present study reveals an additional mechanism for the regulation of PSD-95 by tyrosine phosphorylation. This mechanism may be of pathological significance since it is associated with excitotoxicity in the ischaemic brain.     

Kainate-induced excitotoxicity is dependent upon extracellular potassium concentrations that regulate the activity of AMPA/KA type glutamate receptors

In addition to well-known N-methyl-d-aspartate (NMDA) receptor-mediated excitotoxicity, recent studies suggest that non-NMDA type ionotropic glutamate receptors are also important mediators of excitotoxic neuronal death, and that their functional expression can be regulated by the cellular environment. In this study, we used cerebellar granule cells (CGCs) in culture to investigate kainate (KA)-induced excitotoxicity. Although previous reports indicated that KA induces apoptosis of CGCs in culture, no KA-induced excitotoxic cell death was observed in CGCs treated with KA when cells were maintained in high potassium media (24 mm K+). In contrast, when mature CGCs were shifted into low potassium media (3 mm K+), KA produced significant excitotoxicity. In electrophysiological studies, the KA-induced inward current density was significantly elevated in CGCs shifted into low K+ media compared with those maintained in high K+ media. Non-desensitizing aspects of KA currents observed in this study suggest that these responses were mediated by AMPA rather than KA receptors. In immunofluorescence studies, the surface expression of GluR1 subunits increased when mature CGCs were shifted into a low K+ environment. This study suggests that KA-induced excitotoxicity in mature CGCs is dependent upon the extracellular potassium concentration, which modulates functional expression and excitability of AMPA/KA receptors.     

Knockdown of the aryl hydrocarbon receptor attenuates excitotoxicity and enhances NMDA-induced BDNF expression in cortical neurons

NMDA receptors play dual and opposing roles in neuronal survival by mediating the activity-dependent neurotrophic signaling and excitotoxic cell death via synaptic and extrasynaptic receptors, respectively. In this study, we demonstrate that the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor, is involved in the expression and the opposing activities of NMDA receptors. In primary cultured cortical neurons, we found that NMDA excitotoxicity is significantly enhanced by an AhR agonist 2,3,7,8-tetrachlorodibenzo- p -dioxin, and AhR knockdown with small interfering RNA significantly reduces NMDA excitotoxicity. AhR knockdown also significantly reduces NMDA-increases intracellular calcium concentration, NMDA receptor expression and surface presentation, and moderately decreases the NMDA receptor-mediated spontaneous as well as miniature excitatory post-synaptic currents. However, AhR knockdown significantly enhances the bath NMDA application– but not synaptic NMDA receptor-induced brain-derived neurotrophic factor (BDNF) gene expression, and activating AhR reduces the bath NMDA-induced BDNF expression. Furthermore, AhR knockdown reveals the calcium dependency of NMDA-induced BDNF expression and the binding activity of cAMP-responsive element binding protein (CREB) and its calcium-dependent coactivator CREB binding protein (CBP) to the BDNF promoter upon NMDA treatment. Together, our results suggest that AhR opposingly regulates NMDA receptor-mediated excitotoxicity and neurotrophism possibly by differentially regulating the expression of synaptic and extrasynaptic NMDA receptors.     

Excitotoxicity induced by enhanced excitatory neurotransmission in cultured hippocampal pyramidal neurons.

Cultures of rat hippocampal pyramidal neurons were used to examine the roles of excitatory synaptic transmission, NMDA receptors, and elevated [Ca2+]i in the production of excitotoxicity. In integral of 70% of the cells observed, perfusion with Mg2(+)-free, glycine-supplemented medium induced large spontaneous fluctuations or maintained plateaus of [Ca2+]i. [Ca2+]i fluctuations could be blocked by tetrodotoxin, NMDA receptor antagonists, dihydropyridines, or compounds that inhibit synaptic transmission in the hippocampus, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. When cells were treated with Mg2(+)-free, glycine-supplemented medium and examined 24 hr later, integral of 30% of the neurons were found to have died. Cell death could be inhibited by the same agents that reduced [Ca2+]i fluctuations. These results support a role for direct excitatory synaptic transmission, as opposed to the general release of glutamate, in excitotoxicity. A major role for synaptically activated NMDA receptors, rather than kainate/quisqualate receptors, is also indicated. Neuronal death may be produced by abnormal changes in neuronal [Ca2+]i.     

Neural cell adhesion molecule-associated polysialic acid inhibits NR2B-containing N-methyl-D-aspartate receptors and prevents glutamate-induced cell death

The neural cell adhesion molecule (NCAM) and its associated glycan polysialic acid play important roles in the development of the nervous system and N-methyl-D-aspartate(NMDA)receptor-dependent synaptic plasticity in the adult. Here, we investigated the influence of polysialic acid on NMDA receptor activity. We found that glutamate-elicited NMDA receptor currents in cultured hippocampal neurons were reduced by approximately 30% with the application of polysialic acid or polysialylated NCAM but not by the sialic acid monomer, chondroitin sulfate, or non-polysialylated NCAM. Polysialic acid inhibited NMDA receptor currents elicited by 3 microm glutamate but not by 30 microm glutamate, suggesting that polysialic acid acts as a competitive antagonist, possibly at the glutamate binding site. The polysialic acid induced effects were mimicked and fully occluded by the NR2B subunit specific antagonist, ifenprodil. Recordings from single synaptosomal NMDA receptors reconstituted in lipid bilayers revealed that polysialic acid reduced open probability but not the conductance of NR2B-containing NMDA receptors in a polysialic acid and glutamate concentration-dependent manner. The activity of single NR2B-lacking synaptosomal NMDA receptors was not affected by polysialic acid. Application of polysialic acid to hippocampal cultures reduced excitotoxic cell death induced by low micromolar concentration of glutamate via activation of NR2B-containing NMDA receptors, whereas enzymatic removal of polysialic acid resulted in increased cell death that occluded glutamate-induced excitotoxicity. These observations indicate that the cell adhesion molecule-associated glycan polysialic acid is able to prevent excitotoxicity via inhibition of NR2B subunit-containing NMDA receptors.     

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca²⁺ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca²⁺ channels. Ca²⁺ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca²⁺ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca²⁺.     

Brain-derived neurotrophic factor prevents changes in Bcl-2 family members and caspase-3 activation induced by excitotoxicity in the striatum

Brain-derived neurotrophic factor (BDNF) prevents the loss of striatal neurons caused by excitotoxicity. We examined whether these neuroprotective effects are mediated by changes in the regulation of Bcl-2 family members. We first analyzed the involvement of the phosphatidylinositol 3-kinase/Akt pathway in this regulation, showing a reduction in phosphorylated Akt (p-Akt) levels after both quinolinate (QUIN, an NMDA receptor agonist) and kainate (KA, a non-NMDA receptor agonist) intrastriatal injection. Our results also show that Bcl-2, Bcl-x(L) and Bax protein levels and heterodimerization are selectively regulated by NMDA and non-NMDA receptor stimulation. Striatal cell death induced by QUIN is mediated by an increase in Bax and a decrease in Bcl-2 protein levels, leading to reduced levels of Bax:Bcl-2 heterodimers. In contrast, changes in Bax protein levels are not required for KA-induced apoptotic cell death, but decreased levels of both Bax:Bcl-2 and Bax:Bcl-x(L) heterodimer levels are necessary. Furthermore, QUIN and KA injection activated caspase-3. Intrastriatal grafting of a BDNF-secreting cell line counter-regulated p-AKT, Bcl-2, Bcl-x(L) and Bax protein levels, prevented changes in the heterodimerization between Bax and pro-survival proteins, and blocked caspase-3 activation induced by excitotoxicity. These results provide a possible mechanism to explain the anti-apoptotic effect of BDNF against to excitotoxicity in the striatum through the regulation of Bcl-2 family members, which is probably mediated by Akt activation.     

Selective enhancement of tonic inhibition by increasing ambient GABA is insufficient to suppress excitotoxicity in hippocampal neurons

Gamma-aminobutyric acid (GABA) activates synaptic GABA(A) receptors to generate inhibitory postsynaptic potentials. GABA also acts on extrasynaptic GABA(A) receptors, resulting in tonic inhibition. The physiological role of tonic inhibition, however, remains elusive. We explored the neurophysiological significance of tonic inhibition by testing whether selective activation of extrasynaptic GABA(A) receptors is sufficient to curb excitotoxicity. Tonic inhibition was selectively enhanced by increasing ambient GABA. In both acute hippocampal slices and cultured hippocampal neurons, boosting tonic inhibition alone is insufficient to withstand the hyper-excitability of hippocampal neurons induced by low-magnesium (Mg2+) baths. Furthermore, selective activation of extrasynaptic GABA(A) receptors resulted in no significant neuroprotective effects against glutamate or low-Mg2+-induced neuronal cell deaths. These data imply that under physiological conditions extrasynaptic GABA(A) receptors are optimally activated by ambient GABA and that a further increase in extracellular GABA concentration will not significantly enhance the effect of tonic inhibition on neuronal excitability.