Excitatory Amino Acid Neurotoxicity Research Articles

Excitatory amino acid neurotoxicity at the N-methyl- d-aspartate receptor in cultured neurons: role of the voltage-dependent magnesium block

Results of the present report show that cerebellar neurons in primary culture are resistant to glutamate concentrations as high as 5 mM in the presence of glucose and Mg 2+, but sensitive to glutamate concentrations lower than 35 μM when the neurons are deprived of glucose. Glutamate toxicity is also potentiated when Mg 2+ is removed but glucose and EDTA are present; in this case, higher concentrations of glutamate (1 mM) are required for full toxicity. Glucose concentrations as low as 50 μM are fully protective against the toxicity of 100 μM glutamate; pyruvate and, to a lesser extent, lactate are also protective. Significantly, increasing concentrations of extracellular Mg 2+ are fully protective against the toxicity of 100 μM glutamate in the absence of glucose and against the toxicity of 1 mM glutamate in the presence of glucose and EDTA. We interpret these results as support for our hypothesis that the pivotal event in glutamate's transition to neurotoxin is relief of the Mg 2+ block of the N-methyl- d-aspartate (NMDA) receptor channel, which is known to be voltage-dependent. Partial depolarization in response to depletion of high-energy phosphates relieves the voltage-dependent block enabling glutamate to stimulate an excessive ion influx which results in the death of the neuron by a mechanism which is not yet understood. We propose that this mechanism may be operative in the neuronal damage associated with a variety of neurodegenerative disorders.

The role of neuronal energy in the neurotoxicity of excitatory amino acids

Excitatory amino acids, acting at receptors such as the N-methyl-D-aspartate (NMDA) subtype, are good candidates for a major role in the neuronal death characteristic of Alzheimer's disease. Recent evidence from studies with cultured neurons suggests that perturbations in the energy metabolism of the neuron may be involved in the transition of NMDA agonists from neurotransmitters to neurotoxins via a mechanism that involves relief of the voltage-dependent Mg ++ block of the NMDA channel.

Calpain I activation is specifically related to excitatory amino acid induction of hippocampal damage

Sustained stimulation of receptors for excitatory amino acids leads to both activation of the calcium-dependent cysteine protease calpain I and to the death of receptive neurons. Here, we have examined the relationship between the calpain I activation and neurodegeneration. Calpain I activation was manifested as increased levels of the major proteolytic fragments of the calpain substrate spectrin, detected and quantified by immunoblotting. Intraventricular administration of the excitatory amino acids kainate or N-methyl-D-aspartate (NMDA) produced calpain I-mediated spectrin degradation and hippocampal neuronal loss. The NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid selectively blocked NMDA- but not kainate-induced protease activation and hippocampal damage. Temporally, spectrin degradation preceded the onset of pyramidal cell degeneration monitored by silver-impregnation histochemistry. Only those doses of kainate (0.15-1 microgram) or NMDA (40-80 micrograms) sufficient to cause hippocampal damage markedly increased spectrin breakdown. Both the neuronal damage and calpain I activation induced by kainate occurred primarily in area CA3. Degeneration of hippocampal neurons evoked by colchicine was not accompanied by calpain activation, indicating that proteolysis is not stimulated simply as a secondary response to neuronal destruction. Thus, a close correspondence exists between excitatory amino acid induction of neuronal degeneration and of calpain I-mediated spectrin degradation. The results suggest that calpain I may be an intracellular mediator of excitatory amino acid action, and further, they support the hypothesis that calcium influx and calpain I activation are obligatory events in the initiation of excitatory amino acid neurotoxicity.

Zinc alters excitatory amino acid neurotoxicity on cortical neurons

Recent studies have suggested that large amounts of free zinc may be coreleased during excitatory synaptic transmission at glutamatergic synapses, and may act postsynaptically to decrease actions mediated by N-methyl-D-aspartate (NMDA) receptors, while often increasing neuroexcitation mediated by quisqualate receptors. The present study examined the ability of zinc to alter excitatory amino acid (EAA) neurotoxicity. Murine cortical cell cultures were exposed to EAAs for 5 min in defined solutions, and neuronal cell injury was examined the following day both morphologically and by lactate dehydrogenase assay. Inclusion of 30-500 microM zinc in the exposure solution produced a zinc concentration-dependent, noncompetitive attenuation of NMDA-induced neuronal injury, with an ED50 of about 80 microM. In contrast, zinc produced the same concentration-dependent potentiation of quisqualate neurotoxicity; and with 500 microM zinc, a small potentiation of kainate neurotoxicity was suggested. The effect of zinc on the neurotoxicity of the broad-spectrum agonist glutamate was consistent with these effects on specific agonists, as well as with a previous study showing that glutamate neurotoxicity normally depends predominantly on NMDA-receptor activation. Zinc produced a concentration-dependent reduction in glutamate-induced neuronal injury in a fashion similar to that seen with NMDA, but less effectively. In addition, despite this overall protective effect, zinc paradoxically increased the glutamate-induced destruction of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d)-containing neurons, a subpopulation that was shown in the preceding paper (Koh and Choi, 1988) to exhibit resistance to NMDA receptor-mediated neurotoxicity, and vulnerability to non-NMDA receptor-mediated neurotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Delayed neurotoxicity of excitatory amino acids In vitro

The acute neurotoxicity produced by glutamate and related excitatory amino acids is probably caused by depolarization leading to excessive anionic and cationic fluxes and osmotic lysis. Recently, a more delayed form of glutamate neurotoxicity, which is critically dependent upon calcium influx, has been described in cultured neocortex. We investigated this phenomenon in cultures of dispersed rat hippocampal neurons. When these cultures were briefly incubated with various excitatory amino acids in low extracellular chloride, there was no acute toxicity, but a gradual drop-out of neurons occurred over the next day. When calcium was removed from the extracellular medium during amino acid incubation, this late neuronal loss was not seen. Interestingly, blocking excitatory amino acid receptors in cultures after the amino acid exposure also prevented this delayed neuronal death. In addition, these treated cultures contained neurons with normal physiological properties, and had concentrations of adenosine triphosphate that were close to control values. The findings suggest an amino acid-induced calcium influx may elevate the release of endogenous excitatory transmitter, likely glutamate, and/or increase the sensitivity of these neurons to glutamate. These in vitro observations may partially explain the delayed neuronal loss seen in some pathological conditions affecting man.

The neurotoxicity of excitatory amino acids is produced by passive chloride influx

In the 15 years since the neurotoxic properties of glutamate and related amino acids were first described, there has been no thoroughly convincing explanation of the pathophysiology of excitatory amino acid-induced neuronal death. These substances depolarize central neurons, increase the frequency of neuronal discharge, and augment synaptic activity, leading to the suggestion that one or more of these properties may in some way be responsible for toxicity. More recently, an excessive calcium influx triggered by amino acids has been implicated in this process. As isolation of the different factors potentially involved in amino acid neurotoxicity is virtually impossible in vivo, dispersed hippocampal cultures were used to define the pathophysiology of this process in vitro. The toxicity of glutamate, N-methyl-D-aspartate, and kainate was unaffected when calcium was deleted and tetrodotoxin added to the balanced salt solution bathing the cultures. In parallel experiments, the calcium ionophore A23187 was not toxic in the presence of calcium. These experiments failed to confirm a role for neuronal activity or calcium influx in this process. However, when depolarization was blocked by deleting sodium from the control salt solution, neither glutamate, N-methyl-D-aspartate, nor kainate produced obvious changes. Alternately, when passive chloride influx was prevented by largely deleting chloride from the bath, the cells were also unchanged by the amino acids. Further experiments showed that depolarization produced by high external potassium concentrations or veratridine was also toxic, but only in the presence of external chloride. These experiments suggest that the pathophysiology of amino acid neurotoxicity may be rather straightforward.(ABSTRACT TRUNCATED AT 250 WORDS)