Glutamate receptor and transporter modifications in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation: the contribution of cyclooxygenase-2.

Meloxicam is a non-steroidal anti-inflammatory drug which has been reported to lessen the ischemic transcriptional effects in some of the glutamatergic system genes as well as to decrease the infarct volume in in vivo assays. In this study, we show how the presence of meloxicam decreases cell mortality in assays of oxygen-glucose deprivation (OGD) in rat organotypic hippocampal slices culture. Mortality was measured using propidium iodide. Transcript levels of some glutamatergic system genes, including vesicular and membrane glutamate transporters (VGLUT1, VGLUT2, GLAST-1A, GLT-1, and EAAC-1) and some glutamatergic receptor subunits (NMDA receptor, GluN1, GluN2A and GluN2B subunits and AMPA receptor, GluA1 and GluA2 subunits) were measured by real-time PCR (qPCR). The transcription of vesicular glutamate transporters and glutamatergic receptor subunits, but not membrane glutamate transporters, was modified by the presence of meloxicam. The study demonstrates the neuroprotective role of meloxicam in organotypic hippocampal slice cultures and shows how meloxicam is able to selectively increase or decrease the OGD-induced changes in the expression of the different glutamatergic system genes studied here. We suggest that the neuroprotective role of meloxicam could be due to a modification in the balance of the expression of some glutamatergic receptor subunits, leading to a different stoichiometry of receptors such as NMDA or AMPA. Thus, meloxicam would decrease the excitotoxicity induced by OGD.

PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors.

An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100µM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase.

BACKGROUND AND PURPOSES: Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH: Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS: In male rats with tMCAO, HYDAMTIQ (0.1-10 mg·kg(-1)) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS: Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.

Selective PARP-2 inhibitors increase apoptosis in hippocampal slices but protect cortical cells in models of post-ischaemic brain damage.

BACKGROUND AND PURPOSE: Poly(ADP-ribose) polymerases (PARP)-1 and PARP-2 play complementary tasks in the maintenance of genomic integrity, but their role in cell death or survival processes is rather different. A recently described series of selective PARP-2 inhibitors (UPF-1035, UPF-1069) were used to study the role of PARP-1 and PARP-2 in post-ischaemic brain damage. EXPERIMENTAL APPROACH: We evaluated post-ischaemic brain damage in two different in vitro models: rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD) for 20-30 min, a model characterized by apoptosis-like cell death and mouse mixed cortical cell cultures exposed to 60 min OGD, a model in which cells die with mostly necrosis-like features. KEY RESULTS: In organotypic hippocampal slices, PARP-2 inhibition with UPF-1069 (0.01-1 micromolxL(-1)) caused a concentration-dependent exacerbation (up to 155%) of OGD-induced CA1 pyramidal cell death. Higher concentrations, acting on both PARP-1 and PARP-2, had no effect on OGD injury. In mouse mixed cortical cells exposed to OGD, on the contrary, UPF-1069 (1-10 micromolxL(-1)) significantly reduced post-ischaemic damage. CONCLUSION AND IMPLICATIONS: Selective PARP-2 inhibitors increased post-OGD cell death in a model characterized by loss of neurons through a caspase-dependent, apoptosis-like process (hippocampal slice cultures), but they reduced post-OGD damage and increased cell survival in a model characterized by a necrosis-like process (cortical neurons). UPF-1069 may be a valuable tool to explore the function of PARP-2 in biological systems and to examine the different roles of PARP isoenzymes in the mechanisms of cell death and survival.

Metabotropic glutamate receptors stimulate phospholipase D via different pathways in the adult and neonate rat hippocampus.

In order to characterize the ontogenetic profile of metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) we examined the effects of selected mGlu agents on PLD activity in immature and adult rat hippocampus. The group I mGlu receptor agonist 3,5-dihydroxyphenylglycine stimulated PLD in immature tissue, but reduced the PLD response evoked by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] in adult hippocampus. (2R,1'S,2'R,3'S)-2-(2'-Carboxy-3'-phenylcyclopropyl) glycine (PCCG-13), a recently characterized selective antagonist of PLD-coupled mGlu receptors, displayed a much greater activity in reducing the PLD response to (1S,3R)-ACPD in adult than in neonate hippocampus. Our results lend support to the hypothesis that glutamatergic activation of PLD in the rat hippocampus is developmentally regulated.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

(+)-MCPG induces PKCepsilon translocation in cortical synaptosomes through a PLD-coupled mGluR.

We have tested whether different agonists of metabotropic glutamate receptors could induce translocation of selective protein kinase C isozymes in nerve terminals. In rat cortical synaptosomes 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 100 microM) induced an increase in translocation to 124.6 +/- 5.7% of basal unstimulated conditions of the Ca++-independent protein kinase Cepsilon, but not of the Ca++-dependent isozyme beta. This effect was counteracted by 1-aminoindan-1,5-dicarboxylic acid (100 microM), an antagonist of metabotropic glutamate receptor 1. On the other hand, (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG], an antagonist of metabotropic glutamate receptors group I and II, did not antagonize the effect of 1S,3R-ACPD, and per se induced a translocation of protein kinase Cepsilon of 164 +/- 17.7% of basal unstimulated conditions. Because the (+)-MCPG induction of protein kinase Cepsilon translocation was not antagonized by 1-aminoindan-1, 5-dicarboxylic acid, it is suggested that 1S,3R-ACPD and (+)-MCPG activate this signal transduction pathway through distinct membrane receptors. Indeed (2-[2"-carboxy-3'-phenylcyclopropyl]glycine)-13 (300 nM), a new compound known to antagonize metabotropic glutamate receptors coupled to phospholipase D, was able to antagonize protein kinase Cepsilon translocation induced by (+)-MCPG. Moreover (+)-MCPG directly induced phospholipase D activity, measured as [3H]phosphoethanol production in cortical synaptosomes. These data suggest that in cortical nerve terminals (i) distinct metabotropic glutamate receptors, coupled to different signal transduction pathways, are present, (ii) (+)-MCPG is able to induce protein kinase Cepsilon translocation, and that (iii) a metabotropic glutamate receptor associated to phospholipase D might influence translocation of protein kinase C in a calcium-independent manner.

1-Aminoindan-1,5-dicarboxylic acid and (S)-(+)-2-(3'-carboxybicyclo[1.1.1] pentyl)-glycine, two mGlu1 receptor-preferring antagonists, reduce neuronal death in in vitro and in vivo models of cerebral ischaemia.

Metabotropic glutamate (mGlu) receptors have been implicated in a number of physiological and pathological responses to glutamate, but the exact role of group I mGlu receptors in causing postischaemic injury is not yet clear. In this study, we examined whether the recently-characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) could reduce neuronal death in vitro, following oxygen-glucose deprivation (OGD) in murine cortical cell and rat organotypic hippocampal cultures, and in vivo, after global ischaemia in gerbils. When present in the incubation medium during the OGD insult and the subsequent 24 h recovery period, AIDA and CBPG significantly reduced neuronal death in vitro. The extent of protection was similar to that observed with the nonselective mGlu receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine [(+)MCPG] and with typical ionotropic glutamate (iGlu) receptor antagonists. Neuroprotection was also observed when AIDA or CBPG were added only after the OGD insult was terminated. Neuronal injury was not attenuated by the inactive isomer (-)MCPG, but was significantly enhanced by the nonselective mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid [(1S,3R)-ACPD] and the group I mGlu receptor agonist 3,5-dihydroxyphenylglycine (3,5-DHPG). The antagonists (+)MCPG, AIDA and CBPG were also neuroprotective in vivo, because i. c.v. administration reduced CA1 pyramidal cell degeneration examined 7 days following transient carotid occlusion in gerbils. Our results point to a role of mGlu1 receptors in the pathological mechanisms responsible for postischaemic neuronal death and propose a new target for neuroprotection.

Protection with metabotropic glutamate 1 receptor antagonists in models of ischemic neuronal death: time-course and mechanisms.

In order to study the role of metabotropic glutamate 1 (mGlu1) receptors in ischemic neuronal death, we examined the effects of the recently characterized and relatively selective mGlu1 receptor antagonists 1-aminoindan-1,5-dicarboxylic acid (AIDA) and (S)-(+)-2-(3'-carboxybicyclo[1.1.1]pentyl)-glycine (CBPG) in murine cortical cell cultures and rat organotypic hippocampal slices exposed to oxygen glucose deprivation (OGD) and in vivo, following transient global ischemia in gerbils. AIDA and CBPG significantly reduced neuronal death when added to the incubation medium during the OGD insult and the subsequent recovery period. Neuroprotection was observed even when these compounds were added up to 60 min (in cortical neurons) or 30 min (in hippocampal slices) after OGD. In vivo, i.c.v. administration of AIDA and CBPG reduced hippocampal CA1 pyramidal cell injury following transient global ischemia. Neuroprotection was also observed when AIDA was added to the hippocampal perfusion fluid in microdialysis experiments, and this effect was associated with an increase in the basal output of GABA. These findings demonstrate that AIDA and CBPG are neuroprotective when administered during the maturation of ischemic damage and that different mechanisms are likely to be involved in mediating their effects following blockade of mGlu1 receptors in cortical and hippocampal neurons.

Antagonist pharmacology of metabotropic glutamate receptors coupled to phospholipase D activation in adult rat hippocampus: focus on (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine versus 3, 5-dihydroxyphenylglycine.

Metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) appear to be distinct from any known mGlu receptor subtype linked to phospholipase C or adenylyl cyclase. The availability of antagonists is necessary for understanding the role of these receptors in the central nervous system, but selective ligands have not yet been identified. In a previous report, we observed that 3, 5-dihydroxyphenylglycine (3,5-DHPG) inhibits the PLD response induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in adult rat hippocampal slices. We now show that the antagonist action of 3, 5-DHPG (IC50 = 70 microM) was noncompetitive in nature and nonselective, because the drug was also able to reduce PLD activation elicited by 100 microM norepinephrine and 1 mM histamine. In the search for a selective and more potent antagonist, we examined the effects of sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG) on the PLD-specific transphosphatidylation reaction resulting in the formation of [3H]phosphatidylethanol. The (2R,1'S,2'R,3'S)-PCCG stereoisomer (PCCG-13) antagonized the formation of [3H]phosphatidylethanol induced by 100 microM (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate in a dose-dependent manner and with a much lower IC50 value (25 nM) compared with 3,5-DHPG. In addition, increasing concentrations of PCCG-13 were able to shift to the right the agonist dose-response curve but had no effect when tested on other receptors coupled to PLD. The potent, selective, and competitive antagonist PCCG-13 may represent an important tool for elucidating the role of PLD-coupled mGlu receptors in adult hippocampus.

Neuroprotective effects of kynurenine-3-hydroxylase inhibitors in models of brain ischemia.

The neuroprotective effects of two kynurenine hydroxylase inhibitors, (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfona mide (Ro 61-8048), were studied in vitro and in vivo. In organotypic hippocampal slice cultures deprived of oxygen and glucose, these inhibitors significantly reduced neuronal damage. In gerbils subjected to bilateral carotid occlusion for 5 min, the administration of mNBA (400 mg/kg i.p., 3 times) or Ro 61-8048 (40 mg/kg i.p., 3 times) dramatically decreased the percentage of damaged pyramidal neurones in the hippocampal CA1 region. Finally, in rats with permanent occlusion of the middle cerebral artery, mNBA (200-400 mg/kg i.p.) and Ro 61-8048 (40 mg/kg i.p.) administration reduced the infarct volume. Our results demonstrate that ischemic neuronal damage may be significantly decreased by inhibiting kynurenine hydroxylase.

The GluR2 (GluR-B) hypothesis: Ca(2+)-permeable AMPA receptors in neurological disorders.

The abnormal influx of Ca2+ through glutamate receptor channels is thought to contribute to the loss of neurons associated with a number of brain disorders. Until recently, the NMDA receptor was the only glutamate receptor known to be Ca(2+)-permeable. It is now well established that AMPA receptors exist not only in Ca(2+)-impermeable but also in Ca(2+)-permeable forms. AMPA receptors are encoded by four genes designated gluR1 (gluR-A) through gluR4 (gluR-D). The presence of the gluR2 subunit renders heteromeric AMPA receptor assemblies Ca(2+)-impermeable. Recent studies involving animal models of transient forebrain ischemia and epilepsy show that gluR2 mRNA is downregulated in vulnerable neurons. These observations suggest that downregulation of gluR2 gene expression may serve as a 'molecular switch' leading to the formation of Ca(2+)-permeable AMPA receptors and enhanced toxicity of endogenous glutamate following a neurological insult.

Pharmacological characterization of 1-aminoindan-1,5-dicarboxylic acid, a potent mGluR1 antagonist.

We examined the pharmacological profile of 1-aminoindan-1,5-dicarboxylic acid (AIDA), a rigid (carboxyphenyl)glycine derivative acting on metabotropic glutamate receptors (mGluRs). In cells transfected with mGluR1a, AIDA competitively antagonized the stimulatory responses of glutamate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] on phosphoinositide hydrolysis (pA2 = 4.21). In cells transfected with mGluR5a, AIDA displayed a much weaker antagonist effect. In transfected cells expressing mGluR2, AIDA (< or = 1 mM) did not affect the inhibition of forskolin-stimulated adenylate cyclase activity induced by (1S,3R)-ACPD, but at large concentrations, it displayed a modest agonist activity. In rat hippocampal or striatal slices, AIDA (0.1-1 mM) reduced the effects of (1S,3R)-ACPD on phospholipase C but not on adenylate cyclase responses, whereas (+)-alpha-methyl-4-carboxyphenylglycine (0.3-1 mM) was an antagonist on both transduction systems. In addition, AIDA (0.3-1 mM) had no effect on mGluRs coupled to phospholipase D, whereas (+)-alpha-methyl-4-carboxy-phenylglycine (0.5-1 mM) acted as an agonist with low intrinsic activity. In rat cortical slices, AIDA antagonized the stimulatory (mGluR1-mediated) effect of (1S,3R)-ACPD on the depolarization-induced outflow of D-[3H]aspartate, disclosing an inhibitory effect ascribable to (1S,3R)-ACPD activating mGluR2 and/or mGluR4. Finally, mice treated with AIDA (0.1-10 nmol i.c.v.) had an increased pain threshold and difficulties in initiating a normal ambulatory behavior. Taken together, these data suggest that AIDA is a potent, selective and competitive mGluR1 a antagonist.

Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus.

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.

The GluR2 hypothesis: Ca(++)-permeable AMPA receptors in delayed neurodegeneration.

Increased glutamate-receptor-mediated Ca++ influx is considered an important factor underlying delayed neurodegeneration following ischemia or seizures. Until recently, the NMDA receptor was the only glutamate receptor known to be Ca(++)-permeable. It is now well established that glutamate receptors of the AMPA type, encoded by a gene family designated GluR1-GluR4, exist in both Ca(++)-permeable and Ca(++)-impermeable forms, depending on their subunit composition and degree of RNA editing. Recombinant channels assembled without GluR2 are permeable to Ca++; channels assembled with (edited) GluR2 are Ca(++)-impermeable. AMPA receptors in most adult neurons are hetero-oligomers containing GluR2 subunits, but some neurons have GluR2-less, Ca(++)-permeable receptors. The "GluR2 hypothesis" predicts that a relative reduction in the expression of GluR2 results in enhanced Ca++ influx through newly synthesized AMPA receptors, thereby increasing neurotoxicity of endogenous glutamate. Recent observations indicate reduction in GluR2 expression and predict formation of Ca(++)-permeable AMPA receptors following global ischemia and kainate-induced status epilepticus; these changes are likely to be a major factor contributing to the delayed neurodegeneration that follows these pathological events. The delayed neurodegeneration appears to be primarily apoptotic. Thus, there are at least three strategies for neuroprotection: block of formation of GluR2-less receptors, which may be possible at several levels; block of the GluR2-less receptors themselves; and block of the subsequent apoptosis.

The glycine antagonist and free radical scavenger 7-Cl-thio-kynurenate reduces CA1 ischemic damage in the gerbil.

We examined whether 7-Cl-thio-kynurenate, a potent antagonist at the glycine site of the N-methyl-D-aspartate receptor which also inhibits lipid peroxidation, protected CA1 pyramidal cells following transient forebrain ischemia. Global ischemia was produced in anesthetized gerbils by 5 min bilateral carotid artery occlusion; hippocampal injury was assessed seven days later. 7-Cl-thio-kynurenate (100 mg/kg, i.p. x 5) dramatically attenuated ischemia-induced CA1 cell loss (from 95 +/- 1 to 7 +/- 3%): the protection was associated with a delayed and marked reduction in the animals' temperature. However, when the gerbils were maintained normothermic for at least 360 min, 7-Cl-thio-kynurenate still provided partial (54 +/- 11%) but significant protection. No protection was observed when a reduction in temperature with a time course similar to that caused by 7-Cl-thio-kynurenate was experimentally induced in saline-treated ischemic animals. In situ hybridization revealed that expression of NMDA-R1, a subunit of the N-methyl-D-aspartate receptor, was selectively reduced in CA1 seven days following global ischemia. In ischemic gerbils treated with 7-Cl-thio-kynurenate, protected CA1 cells were still able to express normal amounts of NMDA-R1 messenger RNA. Our results demonstrate that 7-Cl-thio-kynurenate, a glutamate receptor blocker possessing radical scavenger properties, is effective in reducing CA1 hippocampal damage following global ischemia in the gerbil. Since there is growing evidence that a positive feedback interaction between activation of glutamate receptors and free radical formation may be responsible for the generation of ischemic brain damage, drugs capable of interfering with both pathogenic mechanisms may be useful in preventing post-ischemic neuronal death.

AMPA/kainate receptor gene expression in normal and Alzheimer's disease hippocampus.

Alzheimer's disease is a progressive dementia characterized by pronounced degeneration of certain populations of neurons in the hippocampus and cerebral cortex of the brain. One theory is that glutamate receptor-mediated toxicity plays a role in cell loss associated with Alzheimer's disease. We used in situ hybridization to examine GluR1, GluR2, and GluR3 messengerRNAs (encoding alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid/kainate receptor subunits) in sections of autopsy samples of Alzheimer's disease brains and age-, sex-, and post-mortem delay-matched brains from non-demented (control) subjects. GluR1 and GluR2 exhibited a heterogeneous distribution in control brain. GluR1 was expressed in granule cells of the dentate gyrus, in pyramidal cells of the CA1 and CA3 hippocampal subfields and in neurons of the subiculum and entorhinal cortex. GluR2 mRNA was at high density in the dentate gyrus and in CA3, but was at low density in CA1, subiculum, and entorhinal cortex. GluR3 hybridization was at very low levels but selectively localized to the dentate gyrus and CA3. In cerebellum, GluR1 was found in granule and Purkinje cell layers. In sections from Alzheimer's disease brain, a high degree of intersubject variability was observed: some samples showed markedly reduced GluR1 mRNA levels in dentate gyrus, CA1 and CA3 relative to controls; others showed no changes. Microscopic observation of emulsion-dipped sections revealed that the reduction of GluR1 seen in the dentate gyrus and CA3 of some Alzheimer's disease subjects was not due to cell loss.(ABSTRACT TRUNCATED AT 250 WORDS)

The depolarization-induced outflow of D-[3H]aspartate from rat brain slices is modulated by metabotropic glutamate receptors.

Rat brain slices were used to study the effects of different metabotropic glutamate receptor ligands on (i) the depolarization (30 mM KCl)-induced outflow of previously taken up D-[3H]aspartate; (ii) the inhibition of forskolin (30 microM)-induced cyclic AMP accumulation; and (iii) the hydrolysis of phosphoinositides. In addition, the localization of mRNAs coding for different metabotropic glutamate receptor subtypes was detected using in situ hybridization. (1S-3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (30-300 microM), a non selective metabotropic glutamate receptor agonist, significantly increased the KCl-induced output of radioactivity from cortical slices, whereas it inhibited the output from striatal slices. Conversely, (1S,3S,4S)-carboxycyclopropylglycine (0.1-1 microM), a relatively selective agonist of the mGluR2 metabotropic glutamate receptor subtype, had an inhibitory effect on the output of D-[3H]aspartate from both cortical and striatal slices and proved to be the most potent metabotropic glutamate receptor agonist in inhibiting cyclic AMP accumulation, but not in stimulating phosphoinositide hydrolysis. Since 2-amino-4-phosphonobutyrate (a mGluR4, mGluR6 and mGluR7 agonist) was not active in any of the assays tested, we hypothesized that the mGluR2 subtype could be involved in these events. Accordingly, mGluR2 mRNA expression was abundant in cortical neurons projecting to the striatum. Our experiments suggest that the stimulation of metabotropic glutamate receptors may either decrease or increase transmitter release depending on the subtype that prevails in the region under study.