Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including--a) densitometric measurements of the cellular uptake of propidium iodide (PI), --b) histological staining by Flouro-Jade, --c) lactate dehydrogenase (LDH) release to the culture medium, --d) immunostaining for microtubulin-associated protein 2, and --e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of --a) KA and AMPA (and NMDA) in hippocampal slice cultures, and --b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX. We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.     

The developmental expression of fluorescent proteins in organotypic hippocampal slice cultures from transgenic mice and its use in the determination of excitotoxic neurodegeneration

Transgenic mice, expressing fluorescent proteins in neurons and glia, provide new opportunities for real-time microscopic monitoring of degenerative and regenerative structural changes. We have previously validated and compared a number of quantifiable markers for neuronal damage and cell death in organotypic brain slice cultures, such as cellular uptake of propidium iodide (PI), loss of microtubule-associated protein 2 (MAP2), Fluoro-Jade (FJ) cell staining, and the release of cytosolic lactate dehydrogenase (LDH). An important supplement to these markers would be data on corresponding morphological changes, as well as the opportunity to monitor reversible changes or long-term effects in the event of minor damage. As a first step, we present: a) the developmental expression in organotypic hippocampal brain slice cultures of transgenic fluorescent proteins, useful for the visualisation of neuronal subpopulations and astroglial cells; and b) examples of excitotoxic, glutamate receptor-induced degeneration of hippocampal CA1 pyramidal cells, with corresponding astroglial reactivity in such cultures. The slice cultures were set up according to standard techniques, by using one-week old pups from four transgenic mouse strains which express fluorescent proteins in their neurons and/or astroglial cells. From the time of explantation, and subsequently for up to nine weeks in culture, the transgenic neuronal fluorescence displayed the expected characteristics of a developmental, in vivo-like increase, including both the number and localisation of cells, as well as the intensity of fluorescence. At that stage and later, the transgenic fluorescence clearly permitted the visualisation of cell bodies, larger and smaller dendritic branches, spines and axons. In separate experiments, with a 24-hour exposure of matured sliced cultures to 100 microM of the glutamate agonist, N-methyl-D-aspartate (NMDA), we observed, by time-lapse recording, a gradual, but rapid loss of fluorescent CA1 pyramidal cells, accompanied by astrogliosis of transgene fluorescent astroglial cells. Based on these results, we consider that organotypic brain slice cultures from transgenic mice, with fluorescent neurons and glia, combined with detailed visualisation by time-lapse fluorescence microscopy, have great potential for investigating both major irreversible and minor reversible structural changes in neurons and glia, induced by neurotoxins and other neurodegenerative compounds and conditions.     

Long-term, repeated dose in vitro neurotoxicity of the glutamate receptor antagonist L-AP3, demonstrated in rat hippocampal slice cultures by using continuous propidium iodide incubation

Most in vitro models are only used to assess short-term effects of test compounds. However, as demonstrated here, hippocampal slice cultures can be used for long-term studies. The test compound used was the metabotropic glutamate receptor antagonist, L(+)-2-amino-3-phosphonopropionic acid (L-AP3), which is known to be toxic in vivo after subchronic, but not acute, administration. Degenerative effects were monitored by measuring the cellular uptake of propidium iodide (PI; continuously present in the medium) and lactate dehydrogenase (LDH) leakage, and by using a panel of histological stains. Hippocampal slices, derived from 2-3 day old rats and grown for 3 weeks, were subsequently exposed for the next 3 weeks to 0, 10 or 100microM L-AP3, with PI (2microM) in the culture medium. Exposure to 100microM L-AP3 induced severe toxicity after 4-6 days, shown by massive PI uptake, LDH leakage, changes in MAP2 and GFAP immunostaining, and in Nissl and Timm staining. In contrast, 10microM L-AP3 did not induce detectable neuronal degeneration. Treatment with the NMDA receptor antagonist, MK-801, or the AMPA/KA receptor antagonist NBQX, together with 100microM L-AP3, reduced neurodegeneration down to close to control values. It is concluded that continuous incubation of hippocampal slice cultures with PI is technically feasible for use in studies of inducible neuronal degeneration over time.     

Guanine nucleotides inhibit NMDA and kainate-induced neurotoxicity in cultured rat hippocampal and neocortical neurons

Previous evidence suggests that guanine nucleotides can directly inhibit N-methyl-d-aspartate (NMDA) and AMPA/kainate receptors and antagonize a variety of cellular functions elicited by these glutamate receptor agonists. We investigated the possibility that the guanine nucleotides GTP, GDP, and GMP exert a neuroprotective effect on cultured rat hippocampal or neocortical neurons exposed to the excitotoxicants NMDA (30 microM) or kainate (300 microM). On co-application with NMDA all three nucleotides revealed a comparable rescue effect from 100 microM nucleotide concentrations onwards, with a higher inhibitory potential in hippocampal than in neocortical cultures. Similarly, kainate-induced neurotoxicity was inhibited by all three nucleotides but the inhibitory potential was lower than after application of NMDA. Guanosine had no effect on either culture system. GTP and GDP where hydrolyzed by hippocampal and cortical cultures with GMP accumulating in the medium, suggesting that hydrolysis of GTP had no effect on the effective nucleotide concentration. Our results show that GTP, GDP, and GMP inhibit NMDA- and kainate-mediated neurotoxicity in cultured hippocampal and neocortical neurons. They suggest that guanine nucleotides may be candidates for broadly antagonizing glutamate receptor-mediated neurotoxicity.     

Apoptosis and necrosis occurring in excitotoxic cell death in isolated chick embryo retina

Excitotoxic studies using isolated chick embryo retina indicated that such an in vitro model provides a valid tool to characterize the effect of different agonists for subtypes of glutamate ionotropic receptors. In retinas maintained for 24 h in a Krebs medium, after a brief exposure (30 min) to glutamate agonists, we compared the effects produced by NMDA and non-NMDA-agonists, such as kainic acid (KA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Delayed retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium after exposure to the previously named agonists. Although at high concentrations, both KA and AMPA produced more relevant release than NMDA, 7-8% of total retinal LDH was released after exposure to a 50 microM concentration of non-NMDA agonists. These values were similar to those obtained after 100 microM NMDA. In this regard, retinal tissue appeared to be less sensitive to excitotoxicity based on the activation of NMDA receptor subtype. All three agents produced histopathological lesions typical for excitotoxic damage. A delayed form of excitotoxicity observed in retina segments was predominated by necrotic features. However, the activation of apoptotic machinery early during the incubation period subsequent to brief exposure to NMDA (100 microM) was also present. The activation of caspase enzymes was studied by a fluorometric protease activity assay as well as by western blot analysis. Caspase-3-like activity reached the highest value within 3 h of incubation after exposure to excitotoxin, then the level of enzyme activity declined to lower values. As confirmed by a time-related appearance of TUNEL-positive nuclei, apoptotic features appeared to be specific for retina response to NMDA. In contrast, the exposure to a 50 microM concentration of KA or AMPA induced necrotic cell damage which was evident through the incubation, leading to a delayed mechanism of excitotoxicity. These observations provide evidence that in the retinal model, with regard to agonist concentrations and subtype of glutamate receptors, the cascade of events leading to excitotoxicity may result in either apoptotic or necrotic neuronal cell damage.     

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death. Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity. It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.     

Delayed Excitotoxic Neurodegeneration Induced by Excitatory Amino Acid Agonists in Isolated Retina

Abstract: Evidence from in vitro studies suggests that excitotoxic neuronal degeneration can occur by either an acute or delayed mechanism. Studies of the acute mechanism in isolated chick embryo retina using histological methods indicate that this process is rapidly triggered by activation of glutamate receptors of either the N-methyl-d -aspartate (NMDA) or non-NMDA subtypes. The delayed mechanism, studied primarily in cortical and hippocampal cell cultures prepared from embryonic rodent brain, requires activation of NMDA receptors. In these cell culture systems, stimulation of non-NMDA receptors does not rapidly trigger delayed neuronal degeneration, or does so only indirectly, via activation of NMDA receptors secondary to glutamate release. To provide a more valid basis for comparison of these two mechanisms, we have modified the isolated chick embryo retina model to permit studies of delayed as well as acute excitotoxic neurodegeneration. Retinas maintained for 24 h exhibited no morphological or biochemical signs of damage. Retinal damage was assessed by measuring lactate dehydrogenase (LDH) present in the medium at various times after exposure to agonists and normalized to total LDH in each retina. Glutamate exposure (1 mM, 30 min) did not result in LDH release by the end of the exposure period, but LDH was released over the following 24 h. Briefer periods also led to substantial LDH release. Incubation in the presence of NMDA, or the non-NMDA agonists kainate (KA) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), led rapidly to delayed LDH release. NMDA and AMPA were more potent than glutamate, but high concentrations of glutamate led to more LDH release than high concentrations of these agonists. KA was a powerful excitotoxin, providing more LDH release than glutamate, NMDA, or AMPA at every concentration tested. The delayed LDH release induced by glutamate involved activation of both NMDA and non-NMDA receptors, as a combination of receptor-selective antagonists was necessary to provide complete blockade. These results indicate that glutamate, NMDA, AMPA, and KA all cause delayed as well as acute excitotoxic damage in the retina. It is interesting that brief exposure to the non-NMDA receptor agonists, in relatively low concentrations, led to delayed LDH release. This is different than in other in vitro models of delayed excitotoxic neurodegeneration.     

Aluminum, Iron, and Zinc Ions Promote Aggregation of Physiological Concentrations of β-Amyloid Peptide

Abstract: Mechanisms of non-NMDA receptor-mediated excitotoxicity were studied in embryonic rat hippocampal cultures using kainic acid (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) as agonists. Under basal culture conditions, overnight treatment with AMPA resulted in negligible excitotoxicity as assessed by phase-contrast microscopy and measurement of lactate dehydrogenase (LDH) release. In contrast, similar treatment with KA resulted in marked excitotoxic morphologic changes and release of LDH. Cotreatment of cultures with AMPA but not NMDA effectively blocked KA toxicity, suggesting that AMPA-induced rapid desensitization of the AMPA/KA receptor could account for the lack of prominent direct toxicity as well as AMPA's ability to block KA toxicity. To test this hypothesis, cultures were briefly pretreated with 10 μ M cyclothiazide, a drug reported to block desensitization of the AMPA/KA receptor, and then exposed overnight to cyclothiazide plus AMPA and/or KA. Cyclothiazide-treated cultures were now vulnerable to AMPA as well as KA; moreover, AMPA was unable to block KA toxicity completely, suggesting that cyclothiazide impaired AMPA/KA receptor desensitization. These and related studies suggest that a regulatory site may exist on the AMPA/KA receptor that modulates non-NMDA receptor-mediated excitotoxicity.     

Augmentation of serotonin release by sustained exposure to MDMA and methamphetamine in rat organotypic mesencephalic slice cultures containing raphe serotonergic neurons

Several lines of evidence suggest the involvement of the raphe-serotonergic neurons in addiction to psychostimulants and some recreational drugs. In this study, we established rat organotypic mesencephalic slice cultures containing the raphe nuclei and examined the effects of sustained exposure to 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (METH). Immunostaining for tryptophan hydroxylase (TPH) studies revealed that serotonergic neurons were abundant in the slice cultures. Sustained exposure to MDMA and METH (1-1000 microM) for 4 days had little effect on the serotonin tissue content, [(3)H]citalopram binding, or expression/phosphorylation of TPH. Treatment with MDMA or METH for 30 min increased serotonin release in a concentration-dependent manner. Slice cultures were exposed to MDMA for 4 days following a 1-day withdrawal period and then challenged with MDMA (10 microM). Sustained MDMA exposure augmented MDMA-induced serotonin release in a concentration-dependent manner, indicating serotonergic sensitization. Similar serotonergic sensitization was observed for METH. The development of MDMA-induced serotonergic sensitization was attenuated by the NMDA receptor antagonist, MK-801 (10 microM). These results suggest that in mesencephalic slice cultures sustained MDMA or METH exposure induces serotonergic sensitization through activation of NMDA receptors without serotonergic neurotoxicity. The in vitro model system could help to elucidate the mechanisms underlying drug addiction.     

GluR2 knockdown reveals a dissociation between [Ca2+]i surge and neurotoxicity

Reduction in GluR2 subunit expression and subsequent increases in AMPA receptor mediated Ca(2+) currents were postulated to exacerbate glutamate neurotoxicity following seizures or global ischemia. To directly test the effects of shifting the GluR1/GluR2 subunit ratio on excitotoxicity, GluR2 antisense deoxyoligonucleotides (AS-ODNs) were applied to dissociated hippocampal cultures for 1-8 days. The GluR1/GluR2 protein ratio was examined immunohistochemically and by Western blotting. [Ca(2+)](i) concentrations were determined by ratiometric imaging of Fura 2-loaded cells. The cultures were exposed to glutamate, AMPA, NMDA or kainic acid (KA) 3 days after GluR2 knockdown and cell viability was determined 1 day later by MTT reduction assay or Trypan blue exclusion. Although GluR2 AS-ODNs increased the GluR1/GluR2 protein ratio in a time dependent manner, neurons and glia appeared healthy and MTT reduction values were similar to untreated and sense controls. Basal [Ca(2+)](i) levels were unchanged but [Ca(2+)](i) was selectively increased by agonist stimulation of AMPA receptors. Unexpectedly, delayed neurotoxicity was attenuated at saturating doses of glutamate while little difference in cell viability was observed at lower doses or with the other excitotoxins at any concentration. Therefore, there was a dissociation between rises in AMPA receptor-mediated Ca(2+) influx and neurotoxicity despite marked decreases in GluR2 but not GluR1 immunoreactivity. It is proposed that a modification of AMPA receptor stochiometry that raises agonist-stimulated Ca(2+) influx during an excitotoxic insult may have eventual neuroprotective effects.     

Glutamate Neurotoxicity and the Inhibition of Protein Synthesis in the Hippocampal Slice

In some animal models of ischemia, neuronal degeneration can be prevented by the selective antagonism of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype, suggesting that glutamate released during ischemia causes injury by activating NMDA receptors. The rat hippocampal slice preparation was used as an in vitro model to study the pharmacology of glutamate toxicity and investigate why NMDA receptors are critical in ischemic injury. Acute toxicity was assessed by quantifying the inhibition of protein synthesis, which we confirmed by autoradiography to be primarily neuronal. The effect of NMDA was prevented by the specific antagonists MK-801 and ketamine, as well as by the less selective antagonist kynurenic acid. The less selective antagonists kynurenic acid and 6,7-dinitroquinoxaline-2,3-dione antagonized the effects of quisqualate and NMDA. In contrast to previous observations with dissociated neurons in tissue culture, the toxicity of glutamate was unaffected by antagonists, regardless of the glutamate concentration, the duration of exposure, or the presence of magnesium. The high concentration of glutamate required to inhibit protein synthesis and the inability of receptor antagonists to block the effect of glutamate suggest that either glutamate acts through a non-receptor-mediated mechanism, or that the receptor-mediated nature of glutamate effects are masked in the slice preparation, perhaps by the glial uptake of glutamate. The altered physiology induced by ischemia must potentiate the neurotoxicity of glutamate, because we observed with a brain slice preparation that only high concentrations of glutamate caused neurotoxicity in the presence of oxygen and glucose and that these effects were not reversed by glutamate receptor antagonists.     

Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions

Maintenance of low extracellular glutamate ([Glu](O)) preventing excitotoxic cell death requires fast removal of glutamate from the synaptic cleft. This clearance is mainly provided by high affinity sodium-dependent glutamate transporters. These transporters can, however, also be reversed and release glutamate to the extracellular space in situations with energy failure. In this study the cellular localisation of the glutamate transporters GLAST and GLT-1 in organotypic hippocampal slice cultures was studied by immunofluorescence confocal microscopy, under normal culture conditions, and after a simulated ischemic insult, achieved by oxygen and glucose deprivation (OGD). In accordance with in vivo findings, GLAST and GLT-1 were primarily expressed by astrocytes under normal culture conditions, but after OGD some damaged neurons also expressed GLAST and GLT-1. The potential damaging effect of inhibition of the glutamate transporters by DL-threo-beta-benzyloxyaspartate (DL-TBOA) was studied using cellular uptake of propidium iodide (PI) as a quantitative marker for the cell death. Addition of DL-TBOA for 48 h was found to induce significant cell death in all hippocampal regions, with EC(50) values ranging from 38 to 48 microM for the different hippocampal subregions. The cell death was prevented by addition of the glutamate receptor antagonists NBQX and MK-801, together with an otherwise saturating concentration of DL-TBOA (100 microM). Finally, the effect of inhibition of glutamate release, via reverse operating transporters during OGD, was investigated. Addition of a sub-toxic (10 microM) dose of DL-TBOA during OGD, but not during the subsequent 48 h recovery period, significantly reduced the OGD-induced PI uptake. It is concluded: (1) that the cellular expression of the glutamate transporters GLAST and GLT-1 in hippocampal slice cultures in general corresponds to the expression in vivo, (2) that inhibition of the glutamate transporters induces cell death in the slice cultures, and (3) that partial inhibition during simulation of ischemia by OGD protects against the induced PI uptake, most likely by blocking the reverse operating transporters otherwise triggered by the energy failure.     

Novel Glutamate Receptor Antagonists Selectively Protect Against Kainic Acid Neurotoxicity in Cultured Cerebral Cortex Neurons

The effect on excitatory amino acid (EAA)-induced toxicity of two novel non-N-methyl-D-aspartate (non-NMDA) antagonists 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA) and 2-amino-3-[2-(3-hydroxy-5-methyl-isoxazol-4-yl)methyl-5-methyl-3- oxoisoxazolin-4-yl]propionic acid (AMNH) was tested in primary cultures of cerebral cortex neurons. Such cultures provide a useful model for the investigation of the toxicity of EAAs and a convenient screening system for potential neuroprotective activity of pharmacological agents. It was demonstrated that AMNH and AMOA abolished neurotoxicity induced by kainic acid with IC50 values of 62 +/- 10 and 120 +/- 19 microM, respectively. No effect on neuronal damage induced by NMDA or AMPA could be detected.     

β-N-Oxalylamino-L-Alanine Action on Glutamate Receptors

beta-N-Oxalylamino-L-alanine (L-BOAA) is a non-protein excitatory amino acid present in the seed of Lathyrus sativus L. This excitotoxin has been characterized as the causative agent of human neurolathyrism, an upper motor neuron disease producing corticospinal dysfunction from excessive consumption of the lathyrus pea. Previous behavioral, tissue-culture, and in vitro receptor binding investigations revealed that L-BOAA might mediate acute neurotoxicity through quisqualate (QA)-preferring glutamate receptors. The present study demonstrates the stereospecific action of L-BOAA on glutamate receptor binding in whole mouse brain synaptic membranes. L-BOAA was most active in displacing thiocyanate (KSCN)-sensitive specific tritiated (RS)-alpha-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding (i.e., QA receptor) (Ki = 0.76 microM) with a rank-order potency of QA greater than kainate greater than N-methyl-D-aspartate (NMDA). By contrast, the nonneurotoxic D-BOAA isomer (100 microM) was essentially inactive in displacing radioligands for glutamate receptors, except the NMDA site, where it was equipotent with L-BOAA. Scatchard analysis of L-BOAA displacement of specific [3H]AMPA binding indicated competitive antagonism (KD: control, 135 nM; L-BOAA, 265 nM) without a significant change in QA-receptor density, and Hill plots yielded coefficients approaching unity. Differential L-BOAA concentration-dependent decreases in specific [3H]AMPA binding were observed in synaptic membranes, indicating that the neurotoxin was more potent in displacing specific binding from frontal cortex membranes, followed by that for corpus striatum, hippocampus, cerebellum, and spinal cord. (ABSTRACT TRUNCATED AT 250 WORDS)     

Spatiotemporal evidence of apoptosis-mediated ischemic injury in organotypic hippocampal slice cultures

Oxygen-glucose deprivation (OGD) induced neuron-specific cell death in organotypic hippocampal slice cultures. Neuronal death was first evident in the CA1 region 24 h after the injury as assessed by propidium iodide (PI) labeling, and continued to extend to the CA3/4 region up to 72 h. At 6 days post-OGD, PI labeling was weak and diffuse with no clear demarcation of pyknotic nuclei. To characterize biochemical changes produced by OGD, cellular efflux of three key amino acid neurotransmitters was evaluated. OGD elicited large increases in the release of GABA and aspartate (55- and 4.5-fold increase over basal, respectively), while there were no detectable changes in extracellular glutamate levels. In order to ascertain the existence of the synaptic pool of glutamate, sister cultures were treated with sodium azide. This evoked a strong increase in glutamate release, suggesting the intactness of the glutamate system. Further studies revealed a time-dependent activation of caspase 3 following OGD, shown by immunoblot analysis as well as by confocal laser scanning microscopy. While we did not observe the activation of caspases 1, 2, or 8 in our model, the activation of caspase 9 was evident, peaking at 12 h post-OGD. Despite no apparent increase in glutamate release by ischemic slices, treatment with a N-methyl-D-aspartate (NMDA) antagonist or an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonist significantly reduced neuronal death. Furthermore, a pan-caspase inhibitor (zVAD-fmk), but not the caspase 3 inhibitor (DEVD-fmk), provided partial neuroprotection. Inhibition of a Ca(2+)-dependent cysteine protease, calpain, by MDL28170 also elicited partial neuroprotective effects.     

Actions of excitatory amino acids on mesencephalic trigeminal neurons

Mesencephalic trigeminal (MeV) neurons are primary sensory neurons of which the cell soma is located within the brainstem, and is associated with synaptic contacts. In previous studies it has been reported that these cells are resistant to kainic acid excitotoxicity, and have little or no responsiveness to exogenously applied glutamate or selective agonists. In an in vitro slice preparation with intracellular recording, we have found that these cells respond to pressure-applied glutamate, N-methyl-D-aspartic acid (NMDA), kainate (KA), and (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). The kainate and AMPA responses appear to be mediated by different receptors, at least in part, since they exhibit differing sensitivity to an AMPA receptor selective antagonist. The agonists generally evoke larger responses than glutamate and exhibit a long-duration desensitization requiring approximately 10 min for full recovery. Some cross-desensitization between the glutamate agonists is also observed. Mesencephalic trigeminal neurons exhibit high-frequency oscillatory activity during depolarizations that approach threshold potentials, and these could combine with transmitter-induced depolarizations to enhance the excitability of these cells. Previous reports of nonsensitivity to glutamate and to kainate excitotoxicity are attributable to relatively small responses, and to the desensitization expressed by these neurons.     

Quinolinic acid stimulates synaptosomal glutamate release and inhibits glutamate uptake into astrocytes

Quinolinic acid (QA) is an endogenous neurotoxin involved in various neurological diseases, whose action seems to be exerted via glutamatergic receptors. However, the exact mechanism responsible for the neurotoxicity of QA is far from being understood. We have previously reported that QA inhibits vesicular glutamate uptake. In this work, investigating the effects of QA on the glutamatergic system from rat brain, we have demonstrated that QA (from 0.1 to 10mM) had no effect on synaptosomal L-[3H]glutamate uptake. The effect of QA on glutamate release in basal (physiological K+ concentration) or depolarized (40 mM KCl) conditions was evaluated. QA did not alter K+-stimulated glutamate release, but 5 and 10mM QA significantly increased basal glutamate release. The effect of dizolcipine (MK-801), a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor on glutamate release was investigated. MK-801 (5 microM) did not alter glutamate release per se, but completely abolished the QA-induced glutamate release. NMDA (50 microM) also stimulated glutamate release, without altering QA-induced glutamate release, suggesting that QA effects were exerted via NMDA receptors. QA (5 and 10mM) decreased glutamate uptake into astrocyte cell cultures. Enhanced synaptosomal glutamate release, associated with inhibition of glutamate uptake into astrocytes induced by QA could contribute to increase extracellular glutamate concentrations which ultimately lead to overstimulation of the glutamatergic system. These data provide additional evidence that neurotoxicity of QA may be also related to disturbances on the glutamatergic transport system, which could result in the neurological manifestations observed when this organic acid accumulates in the brain.     

The activation of glutamate receptors by kainic acid and domoic acid

The neurotoxins kainic acid and domoic acid are potent agonists at the kainate and alphaamino-5-methyl-3-hydroxyisoxazolone-4-propionate (AMPA) subclasses of ionotropic glutamate receptors. Although it is well established that AMPA receptors mediate fast excitatory synaptic transmission at most excitatory synapses in the central nervous system, the role of the high affinity kainate receptors in synaptic transmission and neurotoxicity is not entirely clear. Kainate and domoate differ from the natural transmitter, L-glutamate, in their mode of activation of glutamate receptors; glutamate elicits rapidly desensitizing responses while the two neurotoxins elicit non-desensitizing or slowly desensitizing responses at AMPA receptors and some kainate receptors. The inability to produce desensitizing currents and the high affinity for AMPA and kainate receptors are undoubtedly important factors in kainate and domoate-mediated neurotoxicity. Mutagenesis studies on cloned glutamate receptors have provided insight into the molecular mechanisms responsible for these unique properties of kainate and domoate.     

Release of γ-Amino[3H]butyric Acid from Cultured Amacrine-Like Neurons Mediated by Different Excitatory Amino Acid Receptors

The release of preaccumulated gamma-amino[3H]butyric acid ([3H]GABA) from putative GABAergic amacrine cells was studied in neuronal monolayer cultures made from embryonic chick retina. Release was specifically stimulated by excitatory amino acid agonists. N-Methyl-D-aspartate (NMDA; EC50, 19.1 +/- 5.0 microM), kainic acid (EC50, 15.6 +/- 2.3 microM), and the presumptive endogenous ligand glutamate (EC50, 3.6 +/- 0.5 microM) showed the same efficacy. Quisqualic acid, although the most potent agonist (EC50, 0.56 +/- 0.12 microM), was only half as efficacious. The time course of [3H]GABA release and autoradiographic visualization of responsive GABA-accumulating cells suggest that approximately 50% of the [3H]GABA-accumulating cells possess no or very low responsiveness to quisqualic acid. Depolarization (56 mM KCl)-induced release was fivefold lower than the maximal effect elicited by excitatory amino acids. Release of [3H]GABA and of endogenous GABA was entirely independent of extracellular Ca2+ but was completely abolished after replacement of Na+ by choline or Li+. The effects of NMDA and low concentrations of glutamate (up to 10 microM) were blocked by 2-amino-5-phosphonovaleric acid, by MK 801, and (in a voltage-dependent manner) by Mg2+. The reduction of NMDA responses by kynurenic acid was reversed by D-serine, and quisqualic acid competitively inhibited kainic acid-evoked release. Our results show that the cultured [3H]GABA-accumulating neurons, which probably represent the in vitro counterparts of GABAergic amacrine cells, express at least two types of excitatory amino acid receptors (of the NMDA and non-NMDA type), both of which can mediate a Ca2(+)-independent but Na2(+)-dependent release of GABA.     

β-Amyloid Neurotoxicity in Human Cortical Culture Is Not Mediated by Excitotoxins

Abstract: β-Amyloid is a metabolic product of the amyloid precursor protein, which accumulates abnormally in senile plaques in the brains of patients with Alzheimer's disease. The neurotoxicity of 0-amyloid has been observed in cell culture and in vivo, but the mechanism of this effect is unclear. In this report, we describe the direct neurotoxicity of β-amyloid in high-density primary cultures of human fetal cortex. In 36-day-old cortical cultures, β-amyloid neurotoxicity was not inhibited by the broad-spectrum excitatory amino acid receptor antagonist kynurenate or the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid under conditions that inhibited glutamate and NMDA neurotoxicity. In 8-day-old cortical cultures, neurons were resistant to glutamate and NMDA toxicity but were still susceptible to β-amyloid neurotoxicity, which was unaffected by excitatory amino acid receptor antagonists. Treatment with β-amyloid caused chronic neurodegenera-tive changes, including neuronal clumping and dystrophic neurites, whereas glutamate treatment caused rapid neuronal swelling and neurite fragmentation. These results suggest that β-amyloid is directly neurotoxic to primary human cortical neurons by a mechanism that does not involve excitatory amino acid receptors.