N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells.

Exposure of cultured cerebellar granule cells to glutamate results in a concentration-dependent (EC50 = 22.7 +/- 0.4 microM) and delayed (24-72 hr) neurotoxicity, which is blocked by the specific N-methyl-D-aspartate (NMDA) receptor antagonists 2-amino-5-phosphovalerate and MK-801 but is unaffected by the non-NMDA receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Although glutamate toxicity in these cells is mediated by the NMDA subtype of glutamate receptor, pretreatment of cerebellar granule cells with subtoxic concentrations of NMDA markedly antagonizes the neurotoxic actions of glutamate, with an IC50 of 55 +/- 4 microM. The neuroprotective effect of NMDA requires a preincubation time of approximately 120 min to be fully manifested and does not require the presence of NMDA during glutamate exposure. These data demonstrate that NMDA receptors mediate both neurotoxicity and neuroprotection in cerebellar granule cells. Among four glutamate receptor agonists tested (NMDA, quisqualate, ibotenate, and kainate), only NMDA was able to provide a robust neuroprotection against glutamate toxicity. Quisqualate was neither neurotoxic nor neuroprotective, whereas ibotenate, which was nontoxic by itself, induced a small degree of neuroprotection. In contrast, kainate, which was neurotoxic to cerebellar granule cells, also provided considerable neuroprotection against glutamate toxicity. Because preincubation of cerebellar granule cells with NMDA fails to alter NMDA receptor-mediated phosphoinositide hydrolysis or the specific binding of [3H]MK-801 to NMDA receptors, it appears that the neuroprotective effects of NMDA are not due to NMDA receptor desensitization.     

Anti-apoptotic effect of memantine against staurosporine- and low-potassium-induced cell death in cerebellar granule cells: a development-dependent effect

Memantine, a NMDA receptor antagonist used in several experimental models of neuronal cell injury, is a neuroprotective agent that can attenuate neuronal apoptosis connected with over-stimulation of NMDA receptors. In the present study, we evaluated the impact of memantine on apoptosis in primary cerebellar granule cell (CGC) cultures at 7 and 12 day in vitro (DIV). Cell death was induced by staurosporine (St, 0.5 μM) or by decreasing the level of potassium in the culture medium (LP, 5 mM KCl). Both treatments induced cell death in CGC with higher cell-damaging effects at 12 DIV and 7 DIV neurons for St and LP, respectively._Memantine (0.1–2 μM) partially attenuated St-induced apoptosis only in 7 DIV CGC as assessed by DNA fragmentation and LDH release, but not caspase-3 activity. During LP-induced apoptosis, memantine decreased LDH release and DNA fragmentation, but not affected caspase-3 activity in 7 and 12 DIV CGC. Interestingly, we found no beneficial effects of other NMDA antagonists, including a competitive antagonist such as AP-5 (100 μM) and an uncompetitive antagonist such as MK-801, (1 μM). In conclusion, our data suggest that the anti-apoptotic effects of memantine in CGC are developmentally regulated and its neuroprotective action occurs through an NMDAR-independent mechanism.     

Neonatal toluene exposure alters agonist and antagonist sensitivity and NR2B subunit expression of NMDA receptors in cultured cerebellar granule neurons.

Toluene has been reported to antagonize the function of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of neonatal toluene exposure on NMDA receptors in primarily cultured cerebellar granule neurons were examined. Sprague-Dawley rats were treated with toluene (0, 200, 500, and 1000 mg/kg, i.p.) from postnatal day (PN) 4 to PN 7. Under toluene-free conditions, Ca2+ signals of cultured neurons in response to glutamate and NMDA were measured for up to 14 days. The expression of NMDA receptor subunits (NR1, NR2A, and NR2B) at 5-14 days in vitro (DIV) were also determined. Neonatal toluene exposure dose-dependently reduced intracellular Ca2+ signals in response to glutamate/glycine and NMDA/glycine in cultured cerebellar granule neurons, and these effects were gradually decreased with time. Such toluene exposure did not influence the inhibition of Mg2+ or MK801 on NMDA-evoked responses, but it decreased the potency of ifenprodil (an NR2B preferring antagonist). The protein levels of NMDA receptor subunit NR2B were consistently reduced by toluene exposure at 5 DIV, but not at 14 DIV. These results demonstrate that neonatal toluene exposure induces long-term but reversible changes in the function and composition of NMDA receptors. Such changes during developmental stages may contribute to the cerebellar dysfunction observed in fetal solvent syndrome.     

Antillatoxin and kalkitoxin, ichthyotoxins from the tropical cyanobacterium Lyngbya majuscula, induce distinct temporal patterns of NMDA receptor-mediated neurotoxicity.

Curacin-A, antillatoxin and kalkitoxin, natural products from the marine cyanobacterium Lyngbya majuscula, were tested for neurotoxicity in primary cultures of rat cerebellar granule neurons. Curacin-A was non-toxic, whereas antillatoxin and kalkitoxin produced concentration-dependent cytotoxicity with LC50 values of 20.1+/-6.4 and 3.86+/-1.91 nM, respectively. Antillatoxin neurotoxicity was produced acutely, whereas kalkitoxin caused a delayed neurotoxic response. The cytotoxicity produced by both antillatoxin and kalkitoxin was prevented by the non-competitive NMDA receptor antagonists dextrorphan and MK-801.     

Mecamylamine prevents neuronal apoptosis induced by glutamate and low potassium via differential anticholinergic-independent mechanisms

Neuronal loss via apoptosis caused by various stimuli may be the fundamental mechanism underlying chronic and acute neurodegenerative diseases. A drug inhibiting neuronal apoptosis may lead to a practical treatment for these diseases. In this study, treatment with mecamylamine, a classical antagonist of nicotinic acetylcholine receptors (nAChRs), prevented neuronal apoptosis induced by 75 microM glutamate and by low potassium (LK) in cerebellar granule neurons (CGNs) with EC(50)s of 35 and 293 microM, respectively. Two other antagonists of nAChRs, dihydro-beta-erythroidine and tubocurarine, failed to inhibit these two kinds of apoptosis. Mecamylamine inhibited the NMDA (30 microM)-evoked current and competed with [(3)H]MK-801. Furthermore, two inhibiters of the c-Jun N-terminal kinase (JNK) pathway prevented LK-induced apoptosis. Mecamylamine reversed the phosphorylation levels of JNK and c-Jun as well as the expression of c-Jun caused by LK in a Western blot assay. In addition, the JNK/c-Jun pathway was not involved in glutamate-induced cell death of CGNs. Our results suggest that mecamylamine prevents glutamate-induced apoptosis by blocking NMDA receptors at the MK-801 site and LK-induced apoptosis by inhibiting the activation of the JNK/c-Jun pathway.     

Effects of paraoxon on neuronal and lymphocytic cholinergic systems

The cholinergic system in lymphocytes is hypothesized to be a key target for neurotoxic organophosphates (OPs). The present study determined the comparative effects of paraoxon, the active metabolite of OP-parathion, which is detected in the human neuroblastoma line, SH-SY5Y, and leukemic T-lymphocytes, MOLT-3, in vitro. Paraoxon induced cytotoxic effects in a dose- and time-dependent manner in both cells. Further, the paraoxon-induced modulatory effects were comparable despite different cell types, including over-expression of N-terminus acetylcholinesterase (N-AChE) protein, a marker of apoptosis, down-regulations of mRNA encoding M1, M2, and M3 muscarinic acetylcholine receptors (mAChRs), and induction in expression of c-Fos gene, an indication of certain mAChR subtype(s) activation. Furthermore, the non-selective cholinergic antagonist atropine partially attenuated the paraoxon-induced N-AChE and c-Fos activations in both types of cells. These results provide initial and additional information that OPs may similarly induce neuro- and immuno-toxic effects through mAChRs activation, and they underline the potential of using lymphocytes for assessing OPs-induced neurotoxicity.     

Protective effect of fangchinoline on cyanide-induced neurotoxicity in cultured rat cerebellar granule cells

The present study was performed to examine the effect of fangchinoline, a bis- benzylisoquinoline alkaloid, which exhibits the characteristics of a Ca2+ channel blocker, on cyanide-induced neurotoxicity using cultured rat cerebellar granule neurons. NaCN produced a concentration-dependent reduction of cell viability, which was blocked by MK-801, an N-methyl-D-aspartate (NMDA) receptor antagonist, verapamil, L-type Ca2+ channel blocker, and L-NAME, a nitric oxide synthase inhibitor. Pretreatment with fangchinoline over a concentration range of 0.1 to 10 microM significantly decreased the NaCN-induced neuronal cell death, glutamate release into medium, and elevation of [Ca2+]i and oxidants generation. These results suggest that fangchinoline may mitigate the harmful effects of cyanide-induced neuronal cell death by interfering with [Ca2+]i influx, due to its function as a Ca2+ channel blocker, and then by inhibiting glutamate release and oxidants generation.     

Enhancement of NMDA-induced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.     

Pathomorphologic effects of N-methyl-D-aspartate antagonists in the rat posterior ingulate/retrosplenial cerebral cortex: A review

This review discusses available information on the neurotoxicity of N-methyl-D-aspartate (NMDA) antagonists in the posterior cingulate/retrosplenial (PC/RS) cortex of rats. NMDA antagonists block the NMDA receptor, a central nervous system ionotropic glutamate receptor, and are neuroprotective since they reduce injury in animal models of ischemia. There is interest in the development and use of NMDA antagonists in treating human cerebrovascular diseases. However, with certain NMDA antagonists, dose-dependent vacuolization of neurons in the neuroanatomically localized PC/RS cortex occurs as a side effect in rats. NMDA antagonists that cause vacuolization also appear to induce heat shock protein expression and heightened glucose metabolism in the same cortical region. Electron microscopy has shown that after treatment with MK-801 (dizocilpine maleate), a prototypic noncompetitive NMDA antagonist, the onset of vacuolization is very rapid. Additional studies with MK-801 have indicated that susceptibility to vacuolization increases between 30 and 90 days of age. Histologic time course studies have demonstrated that as the dose of MK-801 is increased, some vacuolated neurons become necrotic. Necrotic neurons are readily evident by light microscopy in routine preparations. At a given dose of MK-801, neuronal necrosis is more extensive in female rats than male rats. Furthermore, necrosis increases along an anterior to posterior gradient within the susceptible PC/RS cortex. A number of compounds with varied central nervous system (CNS) pharmacologic activity (anticholinergics, GABAmimetics, antipsychotics, and general anesthetics) partially or completely prevent neuronal vacuolization. These data suggest a complex pathogenesis for NMDA antagonist-mediated neurotoxicity and indicate variables which require consideration when designing and interpreting studies with these compounds. Since recent reports have described failure of some NMDA antagonists to produce these side effects, the issues discussed in this review may or may not apply to all NMDA antagonists.     

Imidazoline-induced neuroprotective effects result from blockade of NMDA receptor channels in neuronal cultures

Imidazolines have been shown to be neuroprotective in focal and global ischemia in the rat. However, their mechanism of action is still unclear. We have studied the neuroprotective effects of imidazolines against NMDA-induced neuronal death and hypoxic insult in cerebellar and striatal neuronal cultures. All of the imidazolines tested decreased the NMDA-mediated neurotoxicity in a non-competitive manner. Antazoline was the most effective (IC(50) of 5 microM, maximal neuroprotection reaching 90% at 100 microM). The neuroprotective effects were still present when the imidazolines were applied during the post-insult period. Antazoline, idazoxan and guanabenz also showed neuroprotective effects against hypoxia-induced neuronal death (neuroprotection reaching 95% for antazoline at 100 microM). Antazoline was still active if applied during the reoxygenation period (15% neuroprotection). To determine the mechanism of the neuroprotective effects, the possible interaction of imidazolines with NMDA receptors was studied. Imidazolines dose-dependently and non-competitively inhibited NMDA currents. As found for the neuroprotective effects, antazoline was the most effective imidazoline, with an IC(50) of 4 microM and a maximal inhibition of 90% at 100 microM. This blockade was rapid, reversible and voltage-dependent. We compared these effects to those of the classical non-competitive antagonist of NMDA channels, MK-801. In contrast to imidazolines, blockade of the NMDA current by MK-801 was voltage-independent and reversible only at positive potentials. When co-applied with MK-801, antazoline prevented the long lasting blockade of the NMDA current by MK-801. These results are consistent with the existence of overlapping binding sites for these drugs on the NMDA receptor channel. They indicate that imidazolines exert a strong neuroprotective effect against excitotoxicity and hypoxia in cerebellar and striatal primary neuronal cultures by inhibiting NMDA receptors. Since these effects were non-competitive, imidazolines appear to be interesting new drugs with therapeutic potential.     

Neuronal death signaling by beta-bungarotoxin through the activation of the N-methyl-D-aspartate (NMDA) receptor and L-type calcium channel

The aim of this study was to elucidate the mechanism of the neurotoxic effect of beta-bungarotoxin (beta-BuTX, a snake presynaptic neurotoxin isolated from the venom of Bungarus multicinctus) on cultured cerebellar granule neurons. beta-BuTX exerted a potent, time-dependent, neurotoxic effect on mature granule neurons. Mature neurons, with an abundance of neurite outgrowths, were obtained after 7-8 days in culture. By means of microspectrofluorimetry and fura-2, we measured the intracellular Ca(2+) concentration ([Ca(2+)](i)) and found it to be increased markedly. BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tertrakis(acetoxymethyl ester)], EGTA, MK801 (dizocilpine maleate), and diltiazem prevented not only the elevation of [Ca(2+)](i), but also the beta-BuTX-induced neurotoxic effect. The signaling pathway involved in the elevation of [Ca(2+)](i) in beta-BuTX-induced neurotoxicity was studied. The results obtained indicated that beta-BuTX initially increased the production of reactive oxygen species and subsequently reduced mitochondrial membrane potential and depleted ATP. All of these events in the signaling pathway were blocked by MK801, diltiazem, EGTA, and BAPTA-AM. These findings suggest that the neurotoxic effect of beta-BuTX is mediated, at least in part, by a cascade of events that include the direct or indirect activation of N-methyl-D-aspartate (NMDA) receptors and L-type calcium channels that, in turn, lead to Ca(2+) influx, oxidative stress, mitochondrial dysfunction, and ATP depletion. Therefore, we suggest that this polypeptide neurotoxin, as a result of its high potency and irreversible properties, is a useful tool to elucidate the mechanisms of neurodegenerative diseases.     

Excitotoxic profiles of novel, low-affinity kainate receptor agonists in primary cultures of murine cerebellar granule cells.

The involvement of low-affinity kainate (KA) receptors in neuronal injury was investigated by employing a variety of agonists active at GluR5-7. Their excitotoxic profiles were determined in primary cultures of cerebellar granule cells, which abundantly expressed low-affinity KA receptors, and in the absence of any AMPA receptor-mediated neurotoxicity. Neurotoxicity induced by these compounds was analysed by phase contrast microscopy, a cell viability assay, the TUNEL technique (apoptosis), and by employing propidium iodide (PI; necrosis). All agonists induced concentration-dependent neurotoxicity, with rank order (EC(50) values; microM): (S)-iodowillardiine (IW) 0.2>(2S,4R)-4-methylglutamate (4-MG) 36>(2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434) 46>KA 74>(RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4yl)propanoic acid (ATPA) 88. IW exposure resulted in apoptosis at lower concentrations (<30 microM) and necrosis at higher concentrations, both of which were attenuated by CNQX (50 microM), but not MK-801 (10 microM). ATPA-mediated neurotoxicity was purely apoptotic and was attenuated by the non-NMDA receptor antagonists. Both IW and ATPA induced injury with the morphological characteristics of apoptosis shown by the presence of TUNEL-positive neurones. LY339434-mediated neuronal injury was only attenuated by MK-801 and was necrotic in nature. Similarly, 4-MG (>30 microM) exposure caused necrosis that was partially attenuated by MK-801 (10 microM) and CNQX (50 microM). The patterns of neurotoxicity possessed a complex pharmacological profile, demonstrated an apoptotic-necrotic continuum and were inconsistent with past findings, further outlining the importance of characterizing novel compounds at native receptors. ATPA and to a lesser extent IW appear to be suitable drugs for low-affinity KA receptors. Since toxicity-mediated by low-affinity KA receptors seem likely to contribute to neurodegenerative conditions, our study importantly examines the excitotoxic profile of these novel agonists.     

Suramin inhibits beta-bungarotoxin-induced activation of N-methyl-D-aspartate receptors and cytotoxicity in primary neurons

We demonstrated that beta-bungarotoxin (beta-BuTX), a snake presynaptic neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of beta-BuTX and the cytoprotective agents against beta-BuTX were studied. The neuronal death of cerebellar granule neurons induced by beta-BuTX was manifested with apoptosis and necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of free radicals, decreased mitochondrial membrane potential, and depleted cellular ATP content. MK801 and suramin effectively suppressed these detrimental effects of beta-BuTX. Furthermore, the [3H]MK801 binding was reduced by unlabeled MK801, beta-BuTX, and suramin. Thus, activation of N-methyl-D-aspartate (NMDA) receptors appeared to play a crucial role in the cytotoxic effects following betaBuTX exposure. In conclusion, the novel finding of this study was that a polypeptide beta-BuTX exerted a potent cytotoxic effect through sequential events, including activating NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism. Suramin, clinically used as a trypanocidal agent, was an effective antagonist against beta-BuTX. Data suggest that suramin might have value to detect the possible pathway of certain neuropathological disorders.     

Studies with neuronal cells: From basic studies of mechanisms of neurotoxicity to the prediction of chemical toxicity.

Neurotoxicology considers that chemicals perturb neurological functions by interfering with the structure or function of neural pathways, circuits and systems. Using in vitro methods for neurotoxicity studies should include evaluation of specific targets for the functionalism of the nervous system and general cellular targets. In this review we present the neuronal characteristics of primary cultures of cortical neurons and of cerebellar granule cells and their use in neurotoxicity studies. Primary cultures of cortical neurons are constituted by around 40% of GABAergic neurons, whereas primary cultures of cerebellar granule cells are mainly constituted by glutamatergic neurons. Both cultures express functional GABAA and ionotropic glutamate receptors. We present neurotoxicity studies performed in these cell cultures, where specific neural targets related to GABA and glutamate neurotransmission are evaluated. The effects of convulsant polychlorocycloalkane pesticides on the GABAA, glycine and NMDA receptors points to the GABAA receptor as the neural target that accounts for their in vivo acute toxicity, whereas NMDA disturbance might be relevant for long-term toxicity. Several compounds from a list of reference compounds, whose severe human poisoning result in convulsions, inhibited the GABAA receptor. We also present cell proteomic studies showing that the neurotoxic contaminant methylmercury affect mitochondrial proteins. We conclude that the in vitro assays that have been developed can be useful for their inclusion in an in vitro test battery to predict human toxicity.     

Synthesis and bioactivity of a new class of rigid glutamate analogues. Modulators of the N-methyl-D-aspartate receptor

A variety of derivatives of azetidine-2,4-dicarboxylic acid were synthesized and examined for their ability to stimulate 45Ca2+ uptake in cultures of cerebellar granule cells. Of the compounds tested, the cis-azetidine-2,4-dicarboxylic acid (10f) was found to be the most potent agent in potentiating glutamate, aspartate, or N-methyl-D-aspartate (NMDA) stimulated 45Ca2+ uptake at the NMDA receptor. The mechanism of action of 10f was further investigated in [3H]MK-801 binding assays and [3H]GABA release from cultured embryonic rat forebrain neurons. All of the results from the functional studies of azetidine 10f are consistent with a selectivity of action at the NMDA receptor. Moreover, azetidine 10f appears to exhibit a dual type of action, behaving as a glutamate-like agonist at higher concentrations and as a positive modulator at concentrations below 50 microM.     

Antagonistic action of caffeine against LY294002-induced apoptosis in cerebellar granule neurons

AIM: To study the effect of caffeine on apoptosis induced by inhibition of 1-phosphatidylinositol 3-kinase in cerebellar granule neurons. METHODS: Cerebellar granule neurons culture, agar gel electrophoresis, and stress-activated protein kinase (SAPK)/c-Jun N-terminal protein kinase (JNK) assay kit to measure SAPK/JNK activity. RESULTS: LY294002 evoked apoptosis concentration-dependently in cerebellar granule neurons. But death resulting from LY294002 was prevented by caffeine in a concentration-dependent manner. The survival effect of caffeine was not affected by inhibitors of ryanodine-sensitive Ca2+ release, nor was it inhibited by L-type channel blockers and N-methyl-D-aspartate (NMDA) receptor blocker. In addition, RP-cAMP, H89, and KN62 were not able to inhibit the protective effect of caffeine. Phosphorylation of c-Jun was necessary for the induction of apoptosis induced by LY294002 in cerebellar granule neurons. But caffeine directly inhibited the activation of JNK and decreased phospho-c-Jun in granule neurons. CONCLUSION: Caffeine inhibited the activation of JNK and decreased the phosphorylation of c-Jun to protect granule neurons from LY294002-induced apoptosis.     

MK-801 prevents the post-ischemic cerebral hypoperfusion, but not the dysfunction of the vagal baroreflex in dogs.

Pretreatment with MK-801, a non-competitive N-methyl-D-aspartate (NMDA) antagonist, failed to protect the vagal component of reflex bradycardia from 5-min global cerebral ischemia in dogs under pentobarbital anesthesia. On the other hand, MK-801 completely prevented the development of the post-ischemic cerebral hypoperfusion without affecting the cerebral blood flow in sham-operated animals. The results suggest that NMDA receptors may participate in the development of the secondary disturbance of the cerebral circulation, but are not involved in the post-ischemic dysfunction of the baroreflex system.     

Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate-induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30 min) of energy deprived cells to L-glutamate (1-100 microM) caused a concentration-dependent neurotoxicity, as determined 24 h later by a decrease in the ability of the cells to metabolize 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L-glutamate-induced neurotoxicity (EC50=5 microM) was blocked by the specific NMDA receptor antagonist MK-801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20 microM (EC100) L-glutamate with the rank order (EC50 in microM): antazoline (13)>cirazoline (44)>LSL 61122 [2-styryl-2-imidazoline] (54)>LSL 60101 [2-(2-benzofuranyl) imidazole] (75)>idazoxan (90)>LSL 60129 [2-(1,4-benzodioxan-6-yl)-4,5-dihydroimidazole](101)>RX82 1002 (2-methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]-(+)-MK-801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L-glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]-(+)-MK-801 binding. In HEK-293 cells transfected to express the NR1-1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage- and concentration-dependent block of glutamate-induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10-12 microM at 0 mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate-induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.     

Excitotoxic potential of the cyanotoxin β-methyl-amino-l-alanine (BMAA) in primary human neurons

The toxicity of the cyanobacterial modified amino acid, BMAA, has been described in rat, mouse and leech neurons. Particular emphasis has been placed on the potential ability of BMAA to induce neuronal damage via excitotoxic mechanisms. Here we present data indicating that the effects observed on lower organisms are also evident in a human model. Our data indicates that BMAA induces increased intracellular Ca²⁺ influx, DNA damage, mitochondrial activity, lactate dehydrogenase (LDH) release and generation of reactive oxygen species (ROS). The amelioration of LDH release in the presence of the N-methyl-d-aspartate (NMDA) receptor antagonist MK801 indicates that the neurotoxic effects of BMAA are mediated via NMDA receptor activation. Additionally, we have shown that BMAA induces the expression of neuronal nitric oxide synthase (nNOS) and caspase-3 indicating that it can stimulate apoptosis in human neurons, presumably via activation of NMDA receptors.