E Salinska

NMDA Receptor-Mediated Arachidonic Acid Release In Neurons: Role In Signal Transduction and Pathological Aspects

The N-methyl-D-aspartate (NMDA)-sensitive subtype of glutamate receptor, which gates Ca(2+)-permeable ion channels, is known for its role in learning and memory formation, in the induction of long-term potentiation, and also in seizure activity and neurotoxicity. In primary cultures of cerebellar neurons, agonists of NMDA receptors induce a dose-dependent release of [3H]arachidonic acid ([3H]AA), which is potentiated by activation of the glycine-positive modulatory site and inhibited by NMDA receptor antagonists. NMDA receptor-induced [3H]AA release is inhibited by quinacrine and partially depends on the presence of extracellular calcium. The [3H]AA release is not sensitive, however, to pretreatment with pertussis or cholera toxin, which suggests a Ca(2+)-dependent activation of phospholipase A2 not employing G proteins. Pretreatment of cultures with the natural and semisynthetic sphingolipids GT1b and PKS 3, respectively, inhibits NMDA receptor-mediated [3H]AA release. We also demonstrated glutamate-evoked [3H]AA release from rat hippocampal slices, which is NMDA receptor mediated, calcium dependent and sensitive to quinacrine. Arachidonic acid and its metabolites have been shown to play a role as second messengers and to modulate neuronal activity. Moreover, they are thought to act as transsynaptic modulators in the mechanism of NMDA receptor-induced long-term potentiation in the hippocampus. Their role in ischemic brain pathology has also been postulated. Our experiments on cultured cerebellar granule cells, incubated in a Mg(2+)-free medium deprived of glucose and oxygen, demonstrated a time-dependent stimulation of [3H]AA release. This release was inhibited by antagonists of NMDA receptors and by quinacrine. Stimulation of NMDA-sensitive glutamate receptors and the subsequent calcium-mediated activation of phospholipase A2 may play a role in the in vivo release of arachidonic acid during brain ischemia. This hypothesis is supported by the observation that the enhanced level of thromboxane B2 in the gerbil brain after 5 min of global ischemia is reduced by the systemic application of either the NMDA antagonist MK-801 or the ganglioside GM1.

The mechanism of 1,2,3,4‐tetrahydroisoquinolines neuroprotection: the importance of free radicals scavenging properties and inhibition of glutamate‐induced excitotoxicity

1‐Methyl‐1,2,3,4‐tetrahydroisoquinoline (1MeTIQ), unlike several other tetrahydroisoquinolines, displays neuroprotective properties. To elucidate this action we compared the effects of 1MeTIQ with 1,2,3,4‐tetrahydroisoquinoline (TIQ), a compound sharing many activities with 1MeTIQ (among them reducing free radicals formed during dopamine catabolism), but offering no clear neuroprotection. We found that the compounds similarly inhibit free‐radical generation in an abiotic system, as well as indices of neurotoxicity (caspase‐3 activity and lactate dehydrogenase release) induced by glutamate in mouse embryonic primary cell cultures (a preparation resistant to NMDA toxicity). However, in granular cell cultures obtained from 7‐day‐old rats, 1MeTIQ prevented the glutamate‐induced cell death and 45Ca2+ influx, whereas TIQ did not. This suggested a specific action of 1MeTIQ on NMDA receptors, which was confirmed by the inhibition of [3H]MK‐801 binding by 1MeTIQ. Finally, we demonstrated in an in vivo microdialysis experiment that 1MeTIQ prevents kainate‐induced release of excitatory amino acids from the rat frontal cortex. Our results indicate that 1MeTIQ, in contrast to TIQ, offers a unique and complex mechanism of neuroprotection in which antagonism to the glutamatergic system may play a very important role. The results suggest the potential of 1MeTIQ as a therapeutic agent in various neurodegenarative illnesses of the central nervous system.

Metabotropic glutamate receptors (mGluRs) are involved in early phase of memory formation: possible role of modulation of glutamate release.

Metabotropic glutamate receptors (mGluRs) groups I and II are involved in the cellular processes of long-term potentiation (LTP) and learning and memory formation. I.c.v. injection of the mGluRs agonist 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) can impair memory formation in some types of learning task. The role of mGluRs in neurotransmitters release and production of second messengers has been suggested. The aim of the present study was to determine the effect of i.c.v. administration of the new potent mGluRs agonist ABHxD-I and compare its effect with that of ACPD. We studied the effect of both agonists on acquisition and memory for a one-trial passive avoidance learning task in day-old chicks and on the training related glutamate (Glu) release. ACPD or ABHxD-I (50 nmole per chick, i.c.v. injection) were administered at different times before or after training and chicks were tested at various times after training. Chicks injected with ABHxD-I 30 min before training showed amnesia when tested 30 min or 3h after training. The amnestic effect of ACPD was significant only 30 min after training. Glu release evoked by 70 mM KCl was measured in slices prepared from the IMHV of chick brain isolated from animals injected with either ACPD or ABHxD-I 30 min before training and tested 30 min after training. Glu concentration was measured using HPLC. Both ACPD and ABHxD-I significantly increased Glu release in slices isolated from untrained chicks (30 and 48% compare to control, respectively, P<0.05). Training itself increased Glu release (41% compared to control, P<0.01) and no additional effect of either ACPD or ABHxD-I was observed. These results suggest that mGluRs groups I and II are involved in the early stages of memory formation and that application of either of the studied mGluRs agonists may interfere with that process. The amnestic effect of ABHxD-I seems to be stronger and longer lasting. Although the mechanism of this effect still remains unclear, our results suggest that disregulation of Glu release by mGluR agonists may participate in this process.

Early changes in extracellular amino acids and calcium concentrations in rabbit hippocampus following complete 15-min cerebral ischemia

The effect of cerebral ischemia on extracellular amino acids and calcium content and on the permeability of the blood-brain barrier was studied by in vivo dialysis of rabbit hippocampus. This was combined with physiological and neurophysiological measurements. It was found that immediately after 15-min ischemia extracellular concentrations of glutamate, aspartate and taurine increased 3-, 2- and 6-fold, respectively, whereas a maximal, 7-fold increase of phosphoethanolamine and persistent elevation of glutamate were observed 45 min after ischemia. Extracellular calcium concentration, monitored with 45Ca2+, increased by 10% during the initial phase of ischemia, and decreased to approx. 74% of the basal level 10 min after ischemia. Recovery of extracellular calcium content was not attained until 45 min of recirculation, at which time the first signs of return of bioelectric activity were noted. Increased permeability of the blood-brain barrier to fluoresceine developed immediately after ischemia and persisted up to 2 h of recirculation. The obtained results are discussed in reference to the noted simultaneity of changes in extracellular excitatory amino acids and calcium concentrations and of brain bioelectric activity during and after ischemia. Causal relations between these effects are suggested.

Beneficial effect of nimodipine on metabolic and functional disturbances in rabbit hippocampus following complete cerebral ischemia.

We investigated the effects of intravenous application of nimodipine on the neurophysiologic, biochemical, and morphologic consequences of 15 minutes of global cerebral ischemia in seven rabbits. In vivo dialysis of the hippocampus was used to determine changes in extracellular concentrations of extracellular calcium and amino acids and blood-brain barrier permeability. Ischemia without treatment produced a rapid disappearance of electroencephalographic activity, a decrease in the concentration of extracellular calcium, the release of neuroactive amino acids, and leakage of methionine to the tissue fluid, plus a significant increase of the blood-brain barrier permeability to fluorescein. Except for permeability and electroencephalographic activity, these parameters normalized during 45 minutes of recirculation; permeability and activity failed to normalize completely during 3 hours of recirculation. After 3 hours of recirculation, morphologic changes in the CA1 hippocampal area were observed. Treatment with nimodipine significantly enhanced electroencephalographic activity recovery and normalization during recirculation, reduced the decrease in extracellular calcium concentration, and prevented the increased permeability of the blood-brain barrier. Nimodipine protected the CA1 area from early morphologic changes and reduced leakage of methionine from brain cells. The beneficial cytoprotective effect of nimodipine, probably related to normalization of calcium homeostasis and blood-brain barrier permeability after ischemia, may reflect both vascular and cellular sites of action.

The role of group I metabotropic glutamate receptors in memory consolidation and reconsolidation in the passive avoidance task in 1-day-old chicks

Although reconsolidation of memory after reminder does not seem to be the simple reiteration of the sequential stages occurring during memory consolidation, both phenomena probably employ similar mechanisms including activation of glutamate receptors and protein synthesis. It is known that group I metabotropic glutamate receptors (mGluRs) are involved in memory consolidation and modulation of protein synthesis. The aim of present study was to investigate the role of mGluR5 in memory consolidation and reconsolidation and to determine whether inhibition of these receptors may affect protein synthesis in these processes. The one-trial passive avoidance task on chicks was used as the experimental model of learning. Injection of the mGluR5 antagonist MPEP into a specific chick brain region IMM resulted in amnesia, provided the injection was made either shortly before or after training, or approximately 4 h after training. This amnesia was permanent, resembling the effects of protein synthesis inhibitors. MPEP injection immediately after reminder resulted in only a transient amnesia revealed 1h later. Increased expression of Zif/268 and c-Fos proteins 2 h after initial training was abolished bilaterally in chicks injected with MPEP. Injection of MPEP immediately after reminder did not inhibit c-Fos and Zif/268 expression, on the contrary, their expression was increased, specifically in left IMM and was similar to that observed after initial training. These results show that at least in the chick model mGluR5 play an important role in both consolidation and reconsolidation of memory but the mechanisms triggered by their activation in these processes differ. It is suggested that Ca(2+) signal derived from mGluR5 stimulation is necessary for complete memory consolidation, whereas during reconsolidation other mGluR5 triggered mechanisms of protein synthesis activation and regulation may be involved.

Blockade of N-methyl-d-aspartate-sensitive excitatory amino acid receptors with 2-amino-5-phosphonovalerate reduces ischemia-evoked calcium redistribution in rabbit hippocampus

To evaluate the participation of excitatory amino acid receptors sensitive to N-methyl-D-aspartate (NMDA) in ischemia-evoked redistribution of Ca2+ ions from the extra- to the intracellular compartment of the hippocampus, 2-amino-5-phosphonovalerate (APV), a specific antagonist of NMDA receptors, was administered to the rabbit hippocampus through a dialysis probe before, during, and after complete reversible 15-min cerebral ischemia. Microdialysis of the hippocampus allowed us to determine the changes in extracellular calcium and amino acid concentrations and to monitor the permeability of the blood-brain barrier (BBB) to fluorescein. Moreover, EEG and general physiological parameters were registered. APV significantly reduced the ischemic drop of calcium and increased the taurine and phosphoethanolamine content in the extracellular compartment, whereas changes in concentrations of other amino acids and BBB permeability were not modified. Local administration of APV also improved the recovery of EEG activity after ischemia. Inhibition by APV of ischemia-induced calcium redistribution in the hippocampus suggests a major role of NMDA receptors in the influx of calcium to hippocampal neurons during cerebral ischemia.

Modulation of glutamate transport and receptor binding by glutamate receptor antagonists in EAE rat brain.

The etiology of multiple sclerosis (MS) is currently unknown. However, one potential mechanism involved in the disease may be excitotoxicity. The elevation of glutamate in cerebrospinal fluid, as well as changes in the expression of glutamate receptors (iGluRs and mGluRs) and excitatory amino acid transporters (EAATs), have been observed in the brains of MS patients and animals subjected to experimental autoimmune encephalomyelitis (EAE), which is the predominant animal model used to investigate the pathophysiology of MS. In the present paper, the effects of glutamatergic receptor antagonists, including amantadine, memantine, LY 367583, and MPEP, on glutamate transport, the expression of mRNA of glutamate transporters (EAATs), the kinetic parameters of ligand binding to N-methyl-D-aspartate (NMDA) receptors, and the morphology of nerve endings in EAE rat brains were investigated. The extracellular level of glutamate in the brain is primarily regulated by astrocytic glutamate transporter 1 (GLT-1) and glutamate-aspartate transporter (GLAST). Excess glutamate is taken up from the synaptic space and metabolized by astrocytes. Thus, the extracellular level of glutamate decreases, which protects neurons from excitotoxicity. Our investigations showed changes in the expression of EAAT mRNA, glutamate transport (uptake and release) by synaptosomal and glial plasmalemmal vesicle fractions, and ligand binding to NMDA receptors; these effects were partially reversed after the treatment of EAE rats with the NMDA antagonists amantadine and memantine. The antagonists of group I metabotropic glutamate receptors (mGluRs), including LY 367385 and MPEP, did not exert any effect on the examined parameters. These results suggest that disturbances in these mechanisms may play a role in the processes associated with glutamate excitotoxicity and the progressive brain damage in EAE.

1-Methyl-1,2,3,4-tetrahydroisoquinoline and established uncompetitive NMDA receptor antagonists induce tolerance to excitotoxicity

The aim of this study was to establish the antagonistic effects of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) on NMDA receptors and its neuroprotective abilities on primary cultures of rat cerebellar granule cells exposed for 30 min to 250 or 100 μM glutamate. Neuronal viability was tested after 24 h with propidium iodide or calcein/ethidium homodimer-1 staining. The neuroprotective potential of 100, 250 or 500 μM 1MeTIQ was compared with established uncompetitive NMDA receptor antagonists, 0.5 μM MK-801, or 5 μM memantine. These substances were applied for 30 min either together with glutamate, 24 or 48 h before glutamate, or 0.5 h, 1 h and 3 h after exposure to the excitotoxin. The results demonstrated that MK-801, memantine and 500 μM 1MeTIQ induced an almost complete neuroprotection when co-applied with glutamate, but lower concentrations of 1MeTIQ were slightly less effective. Similar effects for 1MeTIQ and the established NMDA receptor antagonists were observed in the pretreatment experiments, even with a 48-h lag between the application of the tested substances and the excitotoxic challenge. In the post-treatment experiments, MK-801 and memantine and 500 μM 1MeTIQ applied up to 3 h after the exposure to glutamate significantly reduced the excitotoxic lesion, but 1MeTIQ in lower concentrations was ineffective. These results indicate that 1MeTIQ shares neuroprotective abilities with established uncompetitive NMDA receptor antagonists, which suggests that its inhibitory effect on NMDA receptors plays a key role in its anti-excitotoxic activity. Moreover, our data disclose a new mechanism of 1MeTIQ-evoked neuroprotection based on the induction of neuronal tolerance to excitotoxicity.

NMDA-induced 45Ca release in the dentate gyrus of newborn rats: in vivo microdialysis study.

This in vivo study, aimed at detecting the N-methyl-D-aspartate (NMDA) evoked Ca(2+)-induced Ca(2+) release from intracellular stores in the neonatal rat brain, demonstrates that the application of 5 mM N-methyl-D-aspartate via a microdialysis probe for 20 min to the dentate gyrus (DG) of halotane-anesthetized 7 day-old (postnatal day 7, PND 7) rats induces a prolonged decrease in Ca(2+) concentration in an initially calcium-free dialysis medium, indicative of a drop in the extracellular concentration of Ca(2+) and Ca(2+) influx to neurons. In parallel experiments, a huge NMDA-evoked release of 45Ca from the pre-labeled endogenous Ca(2+) pool was observed and interpreted as the expression of intracellular Ca(2+) release. Dantrolene (100 microM) significantly inhibited the NMDA-induced 45Ca release, whereas 250 microM ryanodine exerted an unspecific biphasic effect. Autoradiographic and immunocytochemical detection of ryanodine receptors and calbindin D(28K), respectively, in the hippocampal region of PND 7 rats displayed a pronounced expression of [3H]ryanodine binding sites in the DG, but only a slight immunoreactivity of calbindin D(28K). Plastic changes in neurons or excitotoxic neuronal damage induced by the activation of NMDA receptors are mediated by Ca(2+) signals, resulting from an influx of extracellular Ca(2+), and also in some neurons, from the release of intracellular Ca(2+). Our previous in vivo microdialysis experiments visualized NMDA-evoked 45Ca release in the adult rat dentate gyrus, attributable to Ca(2+)-induced Ca(2+) release from the ryanodine-sensitive pool. An additional role of calbindin in the mechanism of this phenomenon has been suggested. This aspect has not been studied in vivo in newborn rats. Our present results indicate that the release of 45Ca from the prelabeled intracellular, dantrolene-sensitive Ca(2+) pool in the DG neurons of immature rats, most probably representing a phenomenon of Ca(2+)-induced Ca(2+) release, significantly participates in the generation of NMDA receptor-mediated intracellular Ca(2+) signals, whereas the role of calbindin D(28K) in the mechanism of 45Ca release is negligible.