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Peripheral inflammation contributes to minimal hepatic encephalopathy in chronic liver diseases, which could be mediated by neuroinflammation. Neuroinflammation in cerebellum of patients with chronic liver diseases has not been studied in detail. Our aim was to analyze in cerebellum of patients with different grades of liver disease, from mild steatohepatitis to cirrhosis and hepatic encephalopathy: (a) neuronal density in Purkinje and granular layers; (b) microglial activation; (c) astrocyte activation; (d) peripheral lymphocytes infiltration; (e) subtypes of lymphocytes infiltrated. Steatohepatitis was classified as SH1, SH2 and SH3. Patients with SH1 show Th17 and Tfh lymphocytes infiltration in the meninges, microglia activation in the molecular layer and loss of 16 ± 4% of Purkinje and 19 ± 2% of granular neurons. White matter remains unaffected. With the progression of liver disease to worse stages (SH2, SH3, cirrhosis) activation of microglia and astrocytes extends to white matter, Bergman glia is damaged in the molecular layer and there is a further loss of Purkinje neurons. The results reported show that neuroinflammation in cerebellum occurs at early stages of liver disease, even before reaching cirrhosis. Neuroinflammation occurs earlier in the molecular layer than in white matter, and is associated with infiltration of peripheral Th17 and Tfh lymphocytes.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants that accumulate in the food chain and are present in human blood and milk. Children born to mothers exposed to PCBs show cognitive deficits, which are reproduced in rats perinatally exposed to PCBs. It has been proposed that PCB-induced cognitive impairment is due to impairment of the glutamate-nitric oxide (NO)-cGMP pathway. The aim of the present work was to assess whether chronic exposure to the nondioxin-like PCB52, PCB138, or PCB180 alters the function of this pathway in primary cultures of rat cerebellar neurons and to assess whether different PCBs have similar or different mechanisms of action. PCB180 and PCB138 impair the function of the glutamate-NO-cGMP pathway at nanomolar concentrations, and PCB52 impairs the function of the glutamate-NO-cGMP pathway at micromolar concentrations. The mechanisms by which different PCBs impair the function of the glutamate-NO-cGMP pathway are different. Each PCB affects the pathway at more than one step but with different potency and, for some steps, in opposite ways. Exposure to the PCBs alters the basal concentrations of intracellular calcium, NO, and cGMP. The three PCBs increase NO; however, PCB52 and PCB138 increase basal cGMP, while PCB180 decreases it. PCB52 and PCB138 decrease the activation of soluble guanylate cyclase by NO, and PCB180 increases it. Long-term exposure to PCB52, PCB180, or PCB138 reduces the activation of NO synthase and the whole glutamate-NO-cGMP pathway in response to activation of N-methyl-d-aspartate receptors. The EC(50) was 300 nM for PCB52 and 2 nM for PCB138 or PCB180. These results show that chronic exposure to nondioxin like PCBs impairs the function of the glutamate-NO-cGMP pathway in cerebellar neurons by different mechanisms and with different potencies. Impaired function of this pathway would contribute to the cognitive alterations induced by perinatal exposure to PCBs in humans.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants present in human blood and milk. Exposure to PCBs during pregnancy and lactation leads to cognitive impairment in children. Perinatal exposure to PCB 153 or PCB 126 impairs the glutamate-nitric oxide-cGMP pathway in cerebellum in vivo and learning ability in adult rats. The aims of this work were: (1) to assess whether long-term exposure of primary cultures of cerebellar neurons to PCB 153 or PCB 126 reproduces the impairment in the function of the glutamate-nitric oxide-cGMP pathway found in rat cerebellum in vivo; (2) to provide some insight on the steps of the pathway affected by these PCBs; (3) to assess whether the mechanisms of interference of the pathway are different for PCB 126 and PCB 153. Both PCB 153 and PCB 126 increase basal levels of cGMP by different mechanisms. PCB 126 increases the amount of soluble guanylate cyclase while PCB 153 does not. PCB 153 reduces the amount of calmodulin while PCB 126 does not. Also both PCBs impair the function of the glutamate-nitric oxide-cGMP pathway by different mechanisms, PCB 153 impairs nitric oxide-induced activation of soluble guanylate cyclase and increase in cGMP while PCB 126 does not. PCB 126 reduces NMDA-induced increase in calcium while PCB 153 does not. When PCB 153 and PCB 126 exhibit the same effect, PCB 126 was more potent than PCB 153, as occurs in vivo.

Polybrominated diphenylethers (PBDEs) are widely used as flame retardants. Significant amounts of PBDEs are present in the milk of lactating women. The possible neurotoxic effects of PBDEs are not well known. Perinatal exposure to PBDEs affects both motor and cognitive functions by mechanisms that remain unclear. Some types of learning depend on N-methyl-D-aspartate receptor activation, which increases intracellular calcium that binds to calmodulin and activates nitric oxide synthase, increasing nitric oxide formation that activates guanylate cyclase, increasing cGMP formation. Part of this cGMP is released to the extracellular fluid. We studied whether prenatal exposure of rats to PBDE99 alters the function of this glutamate-nitric oxide-cGMP pathway in rat brain in vivo. At 10 weeks of age, rats treated with PBDE99 showed increased function of the glutamate-nitric oxide-cGMP pathway in brain in vivo, as assessed by microdialysis in freely moving rats. The increased function of the pathway was reproduced in primary cultures of cerebellar neurons prepared from rats prenatally exposed to PBDE99 as well as in neurons cultured from normal rats and treated in vitro with PBDE99. Increased calmodulin content and activation of soluble guanylate cyclase by nitric oxide contributed to the increased function of the pathway.

Activation of NMDA receptors leads to activation of cAMP-dependent protein kinase (PKA). The main substrates phosphorylated by PKA following NMDA receptor activation remain unidentified. The aim of this work was to identify a major substrate phosphorylated by PKA following NMDA receptor activation in cerebellar neurones in culture, and to assess whether this phosphorylation may be involved in neuronal death induced by excessive NMDA receptor activation. The main PKA substrate following NMDA receptor activation was identified by MALDI-TOFF fingerprinting as the nuclear protein, matrin 3. PKA-mediated phosphorylation of matrin 3 is followed by its degradation. NMDA receptor activation in rat brain in vivo by ammonia injection also induced PKA-mediated matrin 3 phosphorylation and degradation in brain cell nuclei. Blocking NMDA receptors in brain in vivo with MK-801 reduced basal phosphorylation of matrin 3, suggesting that it is modulated by NMDA receptors. Inhibition of PKA with H-89 prevents NMDA-induced phosphorylation and degradation of matrin 3 as well as neuronal death. These results suggest that PKA-mediated phosphorylation of matrin 3 may serve as a rapid way of transferring information from synapses containing NMDA receptors to neuronal nuclei under physiological conditions, and may contribute to neuronal death under pathological conditions.

Hyperammonemia is responsible for most neurological alterations in patients with hepatic encephalopathy by mechanisms that remain unclear. Hyperammonemia alters phosphorylation of neuronal protein kinase C (PKC) substrates and impairs NMDA receptor-associated signal transduction. The aim of this work was to analyse the effects of hyperammonemia on the amount and intracellular distribution of PKC isoforms and on translocation of each isoform induced by NMDA receptor activation in cerebellar neurons. Chronic hyperammonemia alters differentially the intracellular distribution of PKC isoforms. The amount of all isoforms (except PKC zeta) was reduced (17-50%) in the particulate fraction. The contents of alpha, beta1, and epsilon isoforms decreased similarly in cytosol (65-78%) and membranes (66-83%), whereas gamma, delta, and theta; isoforms increased in cytosol but decreased in membranes, and zeta isoform increased in membranes and decreased in cytosol. Chronic hyperammonemia also affects differentially NMDA-induced translocation of PKC isoforms. NMDA-induced translocation of PKC alpha and beta is prevented by ammonia, whereas PKC gamma, delta, epsilon, or theta; translocation is not affected. Inhibition of phospholipase C did not affect PKC alpha translocation but reduced significantly PKC gamma translocation, indicating that NMDA-induced translocation of PKC alpha is mediated by Ca2+, whereas PKC gamma translocation is mediated by diacylglycerol. Chronic hyperammonemia reduces Ca+2-mediated but not diacylglycerol-mediated translocation of PKC isoforms induced by NMDA.

The signal transduction pathways involved in NMDA receptor modulation by other receptors remain unclear. cAMP could be involved in this modulation. The aim of this work was to analyse the contribution of cAMP to NMDA receptor modulation in cerebellar neurones in culture. Forskolin increases cAMP and results in increased intracellular calcium and cGMP that are prevented by blocking NMDA receptors. Similar effects were induced by two cAMP analogues, indicating that cAMP leads to NMDA receptor activation. It has been reported that phosphorylation of Ser897 of the NR1 subunit of NMDA receptors by cAMP-dependent protein kinase (PKA) activates the receptors. Forskolin increases Ser897 phosphorylation. Neither Ser897 phosphorylation nor cGMP increase induced by forskolin are prevented by four inhibitors of PKA, suggesting that NMDA receptor activation is dependent on cAMP but not on PKA. Inhibition of Akt prevents forskolin-induced phosphorylation of Ser897, suggesting a role for Akt in the mediation of the modulation of NMDA receptors by cAMP. Pituitary adenylate cyclase-activating polypeptide (PACAP) activates its receptors, increasing cAMP and also leading to phosphorylation of Ser897 of NR1 and activation of NMDA receptors. These results indicate that cAMP modulates NMDA receptor in cerebellar neurones and may play a role in NMDA receptor modulation by other receptors.

2,5-Hexanedione is a neurotoxic metabolite of hexane. The mechanisms of its neurotoxicity remain unclear. We assessed whether chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway in primary cultures of cerebellar neurons and/or in the cerebellum of rats. Chronic exposure of cultured cerebellar neurons to 2,5-hexanedione (200 microM) reduced by approximately 50% NMDA-induced formation of cGMP. Activation of soluble guanylate cyclase by nitric oxide was reduced by 46%. This treatment reduced the content of neuronal nitric oxide synthase and soluble guanylate cyclase in neurons by 23 and 20%, respectively. In the cerebellum of rats chronically exposed to 2,5-hexanedione (in the drinking water) NMDA-induced formation of cGMP was reduced by 55% as determined by in vivo brain microdialysis. Activation of soluble guanylate cyclase by nitric oxide was reduced by 65%. The content of neuronal nitric oxide synthase and of soluble guanylate cyclase was reduced by 25 and 21%, respectively, in the cerebellum of these rats. The effects are the same in both systems, indicating that cultured neurons are a good model to study the mechanisms of neurotoxicity of 2,5-hexanedione. These results indicate that chronic exposure to 2,5-hexanedione affects the glutamate-nitric oxide-cGMP pathway at different steps both in cultured neurons and in cerebellum of the animal in vivo. The alteration of this pathway may contribute to the neurotoxic effects of 2,5-hexanedione.

Aluminium (Al) is a neurotoxicant and appears as a possible etiological factor in Alzheimer's disease and other neurological disorders. The mechanisms of Al neurotoxicity are presently unclear but evidence has emerged suggesting that Al accumulation in the brain can alter neuronal signal transduction pathways associated with glutamate receptors. In cerebellar neurons in culture, long term-exposure to Al added 'in vitro' impaired the glutamate-nitric oxide (NO)-cyclic GMP (cGMP) pathway, reducing glutamate-induced activation of NO synthase and NO-induced activation of the cGMP generating enzyme, guanylate cyclase. Prenatal exposure to Al also affected strongly the function of the glutamate-NO-cGMP pathway. In cultured neurons from rats prenatally exposed to Al, we found reduced content of NO synthase and of guanylate cyclase, and a dramatic decrease in the ability of glutamate to increase cGMP formation. Activation of the glutamate-NO-cGMP pathway was also strongly impaired in cerebellum of rats chronically treated with Al, as assessed by in vivo brain microdialysis in freely moving rats. These findings suggest that the impairment of the Glu-NO-cGMP pathway in the brain may be responsible for some of the neurological alterations induced by Al.

Metallothionein (MT)-III, a member of the MT family of metal-binding proteins, is mainly expressed in the CNS and is abundant in glutamatergic neurons. Results in genetically altered mice indicate that MT-III may play neuroprotective roles in the brain, but the mechanisms through which this protein functions have not been elucidated. The aim of this work was to assess whether MT-III is able to prevent glutamate neurotoxicity and to identify the step of the neurotoxic process interfered with by MT-III. Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive activation of glutamate receptors, increased intracellular calcium, and increased nitric oxide. It is shown that MT-III prevented glutamate- and nitric oxide-induced neurotoxicity in a dose-dependent manner, with nearly complete protection at 0.3-1 microgram/ml. MT-III did not prevent the glutamate-induced rise of intracellular calcium level but reduced significantly the nitric oxide-induced formation of cyclic GMP. Circular dichroism analysis revealed that nitric oxide triggers the release of the metals coordinated to the cysteine residues of MT-III, indicative of the S(Cys)-nitrosylation of the protein. Therefore, the present results indicate that MT-III can quench pathological levels of nitric oxide, thus preventing glutamate and nitric oxide neurotoxicity.

The role of cGMP in the mediation of glutamate neurotoxicity remains controversial. Some reports indicate that cGMP mediates glutamate neurotoxicity while others indicate that cGMP is neuroprotective. We have studied the role of cGMP in the mediation of glutamate and nitric oxide neurotoxicity in primary cultures of cerebellar neurons. Inhibition of soluble guanylate cyclase prevents glutamate and nitric oxide neurotoxicity. There is a good correlation between inhibition of cGMP formation and neuroprotection. Moreover 8-Br-cGMP, a cell permeable analog of cGMP, induced neuronal death. These results indicate that increased intracellular cGMP is involved in the mechanism of neurotoxicity. Inhibitors of phosphodiesterase did not increase intracellular cGMP but increased the content of cGMP in the extracellular medium and prevented glutamate neurotoxicity. Moreover, addition of cGMP to the extracellular medium also prevented glutamate neurotoxicity in cerebellar neurons in culture. These results are compatible with a neurotoxic effect of increased intracellular cGMP and a neuroprotective effect of increased extracellular cGMP.

Exposure to aluminum (Al) produces neurotoxic effects in humans. However, the molecular mechanism of Al neurotoxicity remains unknown. Al interferes with glutamatergic neurotransmission and impairs the neuronal glutamate-nitric oxide-cyclic GMP (cGMP) pathway, especially in rats prenatally exposed to Al. The aim of this work was to assess whether Al interferes with processes associated with activation of NMDA receptors and to study the molecular basis for the Al-induced impairment of the glutamate-nitric oxide-cGMP pathway. We used primary cultures of cerebellar neurons prepared from control rats or from rats prenatally exposed to Al. Prenatal exposure to Al prevented glutamate-induced proteolysis of the microtubule-associated protein-2, disaggregation of microtubules, and neuronal death, indicating an impairment of NMDA receptor-associated signal transduction pathways. Prenatal exposure to Al reduced significantly the content of nitric oxide synthase and guanylate cyclase and increased the content of calmodulin both in cultured neurons and in the whole cerebellum. This effect was selective for proteins of the glutamate-nitric oxide-cGMP pathway as the content of mitogen-activated protein kinase and the synthesis of most proteins were not affected by prenatal exposure to Al. The alterations in the expression of proteins of the glutamate-nitric oxide-cGMP pathway could be responsible for some of the neurotoxic effects of Al.

Glutamate neurotoxicity in cerebellar neurons in culture is mediated by excessive production of nitric oxide (NO). We anticipated that 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) could act as a scavenger of NO since it contains a position (C-5) highly activated towards nitration reaction. The aim of this work was to assess whether CR-6 acts as an NO scavenger and prevents glutamate neurotoxicity in cultures of cerebellar neurons. It was shown that CR-6 reduced, in a dose-dependent manner, glutamate-induced formation of cGMP (EC50 approximately 15 microM) and prevented glutamate neurotoxicity. The protection was approximately 50% at 3-10 microM and nearly complete at 100 microM. CR-6 did not prevent glutamate-induced activation of NO synthase, but interfered with the glutamate-NO-cGMP pathway at a later step. CR-6 reduced the formation of cGMP induced by S-nitroso-N-acetylpenicillamine (SNAP), an NO-generating agent, indicating that CR-6 acts as a scavenger of NO in cultured neurons. This was further supported by experiments showing that in neurons treated with CR-6 and glutamate, the 5-nitro derivative of CR-6 was formed, as determined by GC-MS analyses. Moreover, in vitro incubation of CR-6 with SNAP also produced the 5-nitroderivative, thus confirming that CR-6 directly reacts with NO. The results reported indicate that CR-6 acts as an NO scavenger in neurons and prevents glutamate neurotoxicity.

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain., (Copyright 1999 Academic Press.)

The aim of this work was to assess whether ammonia concentrations similar to the increase found in the brain of hyperammonemic rats (100 microM), impair N-methyl-D-aspartate (NMDA) receptor-mediated signal transduction. We first measured glutamate neurotoxicity, which in these neurons is mediated by activation of NMDA receptors, as an initial parameter reflecting activation of NMDA receptor-mediated pathways. Long-term treatment of cultured neurons with ammonia prevents glutamate-induced neuronal death. The EC50 was 20 microM, and at 100 microM the protection was complete. The induction of the protective effect was not immediate, but took several hours. Treatment with 100 microM ammonia did not prevent a glutamate- or NMDA-induced rise of intracellular calcium. Ammonia impaired the glutamate-nitric oxide-cGMP (3',5'-cyclic guanosine monophosphate) pathway in a dose- and time-dependent manner. Glutamate-induced formation of cGMP was reduced by 42%, while activation of nitric oxide synthase was not affected. Ammonia reduced by 31% cGMP formation induced by S-nitroso-N-acetyl-penicillamine (SNAP), a NO-generating agent, confirming that the interference occurs at the level of guanylate cyclase activation by nitric oxide. To assess whether chronic moderate hyperammonemia in vivo also impairs the glutamate-nitric oxide-cGMP pathway, we determined by in vivo brain microdialysis in freely moving rats the formation of cGMP induced by NMDA. In hyperammonemic rats, the formation of cGMP induced by NMDA and SNAP was reduced by ca. 60 and 41%, respectively, indicating that chronic hyperammonemia in the animal in vivo also impairs the glutamate-nitric oxide-cGMP pathway. Impairment of this pathway can contribute to the neurological alterations found in hyperammonemia and hepatic encephalopathy.

Ammonia is a main factor in the pathogenesis of hepatic encephalopathy. We found that acute ammonia toxicity is mediated by activation of NMDA receptors. Chronic moderate hyperammonemia prevents acute ammonia toxicity in rats. Chronic exposure of cultured neurons to 1 mM ammonia leads to impaired response of the NMDA receptor to activation by its agonists (due to decreased protein kinase C-mediated phosphorylation) and prevents glutamate (Glu) neurotoxicity. Compounds that prevent ammonia toxicity in mice (e.g. carnitine) also prevent Glu toxicity in cultured neurons. These compounds did not prevent activation of NMDA receptor or the rise of Ca2+. They interfered with subsequent steps in the toxic process. The protective effect of carnitine is mediated by activation of metabotropic Glu receptors. Agonists of mGluRs, especially of mGluR5, prevent Glu toxicity. Agonists of muscarinic receptors also prevent Glu toxicity and there seems to be an interplay between muscarinic and metabotropic Glu receptors in the protective effect. We have tried to identify intracellular events involved in the process of neuronal death. It is known that the rise of Ca2+ is an essential step. Glu leads to depletion of ATP; some compounds (e.g. carnitine) prevent Glu-induced neuronal death without preventing ATP depletion: additional events are required for neuronal death. Glu induces activation of Na+/K+-ATPase, which could be involved in the toxic process. Inhibitors of protein kinase C, calcineurin or nitric oxide synthase prevent Glu toxicity. Our results indicate that Glu toxicity can be prevented at different steps or by activating receptors coupled to the transduction pathways interfering with the toxic process. Agents acting on these steps could prevent excitotoxicity in vivo in animals.

The aim of this work was to assess whether nicotine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons, to try to identify the receptor mediating the protective effect and to shed light on the step of the neurotoxic process which is prevented by nicotine. It is shown that nicotine prevents glutamate and NMDA neurotoxicity in primary cultures of cerebellar neurons. The protective effect of nicotine is not prevented by atropine, mecamylamine or dihydro-beta-erythroidine, but is slightly prevented by hexamethonium and completely prevented by tubocurarine and alpha-bungarotoxin, indicating that the protective effect is mediated by activation of alpha7 neuronal nicotinic receptors. Moreover, alpha-bungarotoxin potentiates glutamate neurotoxicity, suggesting a tonic prevention of glutamate neurotoxicity by basal activation of nicotinic receptors. Nicotine did not prevent glutamate-induced rise of free intracellular calcium nor depletion of ATP. Nicotine prevents glutamate-induced proteolysis of the microtubule-associated protein MAP-2 and disaggregation of the neuronal microtubular network. The possible mechanism responsible for this prevention is discussed.

Humans are exposed to aluminum from environmental sources and therapeutic treatments. However, aluminum is neurotoxic and is considered a possible etiologic factor in Alzheimer's disease and other neurological disorders. The molecular mechanism of aluminum neurotoxicity is not understood. We tested the effects of aluminum on the glutamate-nitric oxide-cyclic GMP pathway in cultured neurons. Neurons were exposed to 50 microM aluminum in culture medium for short-term (4 h) or long-term (8-14 days) periods, or rats were prenatally exposed, i.e., 3.7% aluminum sulfate in the drinking water, during gestation. Chronic (but not short-term) exposure of neurons to aluminum decreased glutamate-induced activation of nitric oxide synthase by 38% and the formation of cyclic GMP by 77%. The formation of cyclic GMP induced by the nitric oxide-generating agent S-nitroso-N-acetylpenicillamine was reduced by 33%. In neurons from rats prenatally exposed to aluminum but not exposed to it during culture, glutamate-induced formation of cyclic GMP was inhibited by 81%, and activation of nitric oxide synthase was decreased by 85%. The formation of cyclic GMP induced by S-nitroso-N-acetylpenicillamine was not affected. These results indicate that chronic exposure to aluminum impairs glutamate-induced activation of nitric oxide synthase and nitric oxide-induced activation of guanylate cyclase. Impairment of the glutamate-nitric oxide-cyclic GMP pathway in neurons may contribute to aluminum neurotoxicity.

1-Aminocyclopentane-trans-1,3-dicarboxylic acid, an agonist of the metabotropic glutamate receptors 1, 2, 3 and 5, prevents neurotoxicity of glutamate and of N-methyl-D-aspartate in primary cultures of cerebellar neurons. The aim of this work was to assess which of the metabotropic glutamate receptors (mGluRs) is responsible for the protective effect. We tested the protective effects of selective agonists for each type of receptor. It is shown that glutamate and N-methyl-D-aspartate neurotoxicity are prevented by the following compounds: 1-amino-cyclo-pentane-trans-1,3-dicarboxylic acid, agonist of mGluR1, 2, 3 and 5; 3,5-dihydroxyphenylglycine, agonist of mGluR1 and 5; S-4-carboxy-3-hydroxyphenylglycine, agonist of mGluR5 and antagonist of mGluR1; trans-azetidine-2,4-dicarboxylic acid, agonist of mGluR5. Glutamate neurotoxicity is not prevented by (2S,1'S,2'S)-2(2'-carboxycyclopropyl)glycine, an agonist of mGluR2 and mGluR3. Moreover, the protective effect of 1-aminocyclo-pentane-trans-1,3-dicarboxylic acid is prevented by alpha-methyl-4-carboxyphenylglycine, an antagonist of mGluR1 and 5, but not by alpha-methyl-4-tetrazoylphenylglycine, an antagonist of mGluR2 and 3. A protective effect of activation of mGluR1 can not be ruled out because of the limitations imposed by the lack of specificity of the agonists and antagonists currently available. The results shown clearly indicate that activation of mGluR5 prevents glutamate and N-methyl-D-aspartate neurotoxicity in primary cultures of cerebellar neurons.

Carnitine prevents acute ammonia toxicity in animals. We propose that acute ammonia toxicity is mediated by activation of N-methyl-D-aspartate receptors and have shown that carnitine prevents glutamate neurotoxicity. The aim of this work was to assess whether other compounds containing a trimethylamine group are able to prevent ammonia toxicity in mice and/or glutamate toxicity in primary neuronal cultures. It is shown that betaine, trimethylamine-N-oxide, choline, acetylcholine, carbachol and acetylcarnitine prevent ammonia toxicity in mice. They also prevent glutamate but not N-methyl-D-aspartate neurotoxicity. Choline, acetylcholine and acetylcarnitine afford partial (approximately 50%) protection at nanomolar concentrations and nearly complete protection at micromolar concentrations. Trimethylamine-N-oxide, carbachol and betaine afford nearly complete protection at approximately 0.2 mM. The protective effect against glutamate neurotoxicity is prevented by 2-amino-3-phosphonopropionic acid, an antagonist of metabotropic glutamate receptors. Atropine, an antagonist of muscarinic receptors, prevents the protective effect of most of the above compounds against ammonia toxicity in mice and against glutamate toxicity in cultured neurons. These results support the idea that acute ammonia toxicity is mediated by activation of N-methyl-D-aspartate receptors and that glutamate neurotoxicity could be prevented by activating metabotropic glutamate receptors and/or muscarinic receptors.