Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning.

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG.

Tolerance Induced by (S)-3,5-Dihydroxyphenylglycine Postconditioning is Mediated by the PI3K/Akt/GSK3ß Signalling Pathway in an In Vitro Model of Cerebral Ischemia.

Ischemic postconditioning (PostC) is an endogenous neuroprotective strategy for cerebral ischemia induced by low activation of glutamate receptors. We have previously shown that the application of the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) 5 min after 30 min of oxygen and glucose deprivation (OGD) reduces CA1 damage in organotypic hippocampal slices by activating the PI3K-Akt signalling pathway. In order to extend these data, we analysed the production of reactive oxygen species (ROS) and the glycogen synthase kinase 3ß (GSK3ß) signalling pathway. Our results show that DHPG PostC was associated with a reduction in the formation of ROS that is massively increased 24 h after OGD exposure. This reduction was prevented by the PI3K inhibitor LY294002, indicating that there is a link between the PI3K/Akt pathway and the formation of ROS in the protective mechanisms of PostC. DHPG PostC also induces a transient increased in GSK3ß phosphorylation and inactivation that is followed by nuclear accumulation of ß-catenin, that probably lead to the up-regulation of neuroprotective genes. Our results propose GSK3ß as new target for neuroprotection, therefore, we verified that the two GSK3ß inhibitors N-(3-Chloro-4-methylphenyl)-5-(4-nitrophenyl)-1,3,4-oxadiazol-2-amine (TC-G 24) and LiCl are neuroprotective agents in OGD and also can be used as PostC agents.

Prokineticins are neuroprotective in models of cerebral ischemia and ischemic tolerance in vitro.

Bv8/prokineticin 2 (PK2) is a member of a bioactive family of peptides that regulate multiple functions in the CNS including hyperalgesia, neurogenesis, neuronal survival and inflammation. Recent studies have associated PK2 and prokineticin receptors (PKR) with human diseases, but because their role in neuropathology is still debated we examined whether prokineticins exert a protective or deleterious role in models of cerebral ischemia and ischemic tolerance in vitro. In order to mimic cerebral ischemia, we exposed primary murine cortical cell cultures or rat organotypic hippocampal slices to appropriate periods of oxygen-glucose deprivation (OGD), which leads to neuronal damage 24 h later. Ischemic tolerance was induced by exposing hippocampal slices to a preconditioning subtoxic pharmacological stimulus (3 µM NMDA for 1 h) 24 h before the exposure to OGD. Bv8 (10-100 nM) attenuated OGD injury in cortical cultures and hippocampal slices, and the effect was prevented by the PKR antagonist PC7. The development of OGD tolerance was associated with an increase in the expression of PK2, PKR1 and PKR2 mRNA and proteins and was prevented by addition of the antagonist PC7 into the medium during preconditioning. Both Bv8 at protective concentrations and the NMDA preconditioning stimulus promoted the phosphorylation of ERK1/2 and Akt. These findings indicate that the prokineticin system can be up-regulated by a defensive preconditioning subtoxic NMDA stimulus and that PK2 may act as an endogenous neuroprotective factor through the activation of the ERK1/2 and Akt transduction pathways.

Long-term Neuroglial Cocultures as a Brain Aging Model: Hallmarks of Senescence, MicroRNA Expression Profiles, and Comparison With In Vivo Models.

Our purpose was to evaluate long-term neuroglial cocultures as a model for investigating senescence in the nervous system and to assess its similarities with in vivo models. To this aim, we maintained the cultures from 15 days in vitro (mature cultures) up to 27 days in vitro (senescent cultures), measuring senescence-associated, neuronal, dendritic, and astrocytic markers. Whole microRNA expression profiles were compared with those measured in the cortex of 18- and 24-month-old C57Bl/6J aged mice and of transgenic TgCRND8 mice, a model of amyloid-ß deposition. Neuroglial cocultures displayed features of cellular senescence (increased senescence-associated-ß-galactosidase activity, oxidative stress, γ-H2AX expression, IL-6 production, astrogliosis) that were concentration dependently counteracted by the antiaging compound resveratrol (1-5 µM). Among the 1,080 microRNAs analyzed, 335 were downregulated or absent in 27 compared with 15 days in vitro and resveratrol reversed this effect. A substantial overlapping was found between age-associated changes in microRNA expression profiles in vitro and in TgCRND8 mice but not in physiologically aged mice, indicating that this culture model displays more similarities with pathological than physiological brain aging. Our results demonstrate that neuroglial cocultures aged in vitro can be useful for investigating the cellular and molecular mechanisms of brain aging and for preliminary testing of protective compounds.

Interplay between histone acetylation/deacetylation and poly(ADP-ribosyl)ation in the development of ischemic tolerance in vitro.

Ischemic tolerance is an endogenous defense program in which exposure to a subtoxic preconditioning insult results in resistance to a subsequent, otherwise lethal, episode of ischemia. Herein, we evaluated the role of histone acetylation/deacetylation in an in vitro model of preconditioning, using rat organotypic hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to CA1 injury 24 h later: tolerance was induced by exposing the slices to preconditioning bouts of NMDA (3 µM for 60 min) 24 h prior to the toxic OGD challenge. Under these conditions, CA1 damage induced by OGD was reduced. The induction of tolerance was prevented by incubating the slices with HDAC inhibitors. NMDA preconditioning was associated with a mild increase in poly(ADP-ribose) polymerase (PARP) activity that was apparently followed, 3 h later, by a mild increase in histone acetylation. Use of PARP and HDAC inhibitors suggests a possible interaction between PARP and HDAC activities in the development of ischemic tolerance. Finally, both PARP and HDAC inhibitors were able to prevent the increase in pERK1/2 induced by NMDA preconditioning. We propose a model in which mild histone acetylation and PARP activity cooperate in producing a neuroprotective response in the development of ischemic tolerance.

Arrays of microLEDs and astrocytes: biological amplifiers to optogenetically modulate neuronal networks reducing light requirement.

In the modern view of synaptic transmission, astrocytes are no longer confined to the role of merely supportive cells. Although they do not generate action potentials, they nonetheless exhibit electrical activity and can influence surrounding neurons through gliotransmitter release. In this work, we explored whether optogenetic activation of glial cells could act as an amplification mechanism to optical neural stimulation via gliotransmission to the neural network. We studied the modulation of gliotransmission by selective photo-activation of channelrhodopsin-2 (ChR2) and by means of a matrix of individually addressable super-bright microLEDs (µLEDs) with an excitation peak at 470 nm. We combined Ca2+ imaging techniques and concurrent patch-clamp electrophysiology to obtain subsequent glia/neural activity. First, we tested the µLEDs efficacy in stimulating ChR2-transfected astrocyte. ChR2-induced astrocytic current did not desensitize overtime, and was linearly increased and prolonged by increasing µLED irradiance in terms of intensity and surface illumination. Subsequently, ChR2 astrocytic stimulation by broad-field LED illumination with the same spectral profile, increased both glial cells and neuronal calcium transient frequency and sEPSCs suggesting that few ChR2-transfected astrocytes were able to excite surrounding not-ChR2-transfected astrocytes and neurons. Finally, by using the µLEDs array to selectively light stimulate ChR2 positive astrocytes we were able to increase the synaptic activity of single neurons surrounding it. In conclusion, ChR2-transfected astrocytes and µLEDs system were shown to be an amplifier of synaptic activity in mixed corticalneuronal and glial cells culture.

Azithromycin: assessment of intrinsic cytotoxic effects on corneal epithelial cell cultures.

PURPOSE: To compare the cytotoxic effects of preservative-free azithromycin on corneal epithelial cells in vivo with those of preservative-free netilmicin and levofloxacin, and the preservative benzalkonium chloride (BAK). METHODS: Rabbit corneal epithelial cells in vitro were incubated for 15 minutes or 6 hours with commercially available ophthalmic preservative-free netilmicin 0.3%, levofloxacin 0.3%, or azithromycin 1.5% preparations or different concentrations of unpreserved azithromycin and different concentrations of BAK. Qualitative analysis was undertaken using phase-contrast optics to examine the morphological aspects of cell cultures and quantitative analysis was undertaken by measuring the release of the cytoplasmic enzyme lactate dehydrogenase into the medium immediately and 24 hours after exposure to drugs. Finally, we observed the wound-healing rate of mechanically injured corneal epithelial cells exposed to each antibiotic ophthalmic preparation for 48 hours. RESULTS: Our results show that both the commercially available unpreserved mono-dose preparation of azithromycin and ophthalmic preparations of azithromycin up to a concentration of 1.5% were virtually devoid of harmful effects under our experimental conditions. This was not significantly different from the results obtained for the other antibiotic preparations (P > 0.05) tested, but was unlike the results obtained for BAK. Azithromycin 1.5% also showed good recovery properties after a mechanical wound test. CONCLUSION: Under our experimental conditions, unpreserved azithromycin 1.5% showed a much lower toxicity than BAK and did not interfere with the wound-healing process.

Extracellular chaperones prevent Aß42-induced toxicity in rat brains.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid ß (Aß42) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aß oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aß42 oligomers pre-incubated for 1h with two extracellular chaperones, namely clusterin and α2-macroglobulin. The chaperones were found to prevent Aß42-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aß42-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aß42 colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aß42 aggregation, as previously demonstrated, but also by suppressing the toxicity of Aß42 oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.

Amyloid-ß oligomer synaptotoxicity is mimicked by oligomers of the model protein HypF-N.

Protein misfolded oligomers are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-ß peptide play a central role in Alzheimer's disease pathogenesis, being implicated in synaptic dysfunction. Here we show that the oligomers formed by a protein that has no link with human disease, namely the N-terminal domain of HypF from Escherichia coli (HypF-N), are also synaptotoxic. HypF-N oligomers were found to (i) colocalize with post-synaptic densities in primary rat hippocampal neurons; (ii) induce impairment of long-term potentiation in rat hippocampal slices; and (iii) impair spatial learning of rats in the Morris Water Maze test. By contrast, the native protein and control nontoxic oligomers had none of such effects. These results raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of Alzheimer's disease, with advantages over other systems for their stability, reproducibility, and costs. The results also suggest that, in the context of a compromised protein homeostasis resulting from aggregation of the amyloid ß peptide, a number of oligomeric species sharing common synaptotoxic activity can arise and cooperate in the pathogenesis of the disease.

Lipid rafts mediate amyloid-induced calcium dyshomeostasis and oxidative stress in Alzheimer's disease.

Several lines of evidence suggest that the initial events of amyloid-ß peptide (Aß) oligomerization and deposition in Alzheimer's disease (AD) involve the interaction of soluble oligomers with neuronal membranes. In this study, we show that Aß42 oligomers are recruited to lipid rafts, which are ordered membrane microdomains rich in cholesterol and gangliosides, resulting in lipid peroxidation, Ca(2+) dyshomeostasis and membrane permeabilization in primary fibroblasts from familial AD patients (FAD) bearing APPVal717Ile, PS-1Leu392Val or PS-1Met146Leu gene mutations. Moreover, the presence of significantly higher levels of lipid peroxidation correlated with greater structural modification in detergent resistant domains (DRMs) isolated from APP and PS-1 fibroblasts, compared to WT fibroblasts from healthy subjects. Modulation of raft GM1, including modest depletion of GM1 content and interference with GM1 exposure or negative charge, precluded the interaction of amyloid aggregates with the plasma membrane and the resulting cell damage in FAD fibroblasts and rat brains cortical neurons. These findings suggest a specific role for raft domains as primary mediators of amyloid toxicity in AD neurons.

Mild activation of poly(ADP-ribose) polymerase (PARP) is neuroprotective in rat hippocampal slice models of ischemic tolerance.

Ischemic tolerance is a phenomenon in which exposure to a mild preconditioning stress results in resistance to a subsequent lethal ischemic insult. Here we investigated the role of poly(ADP-ribose) polymerase (PARP) in the development of ischemic tolerance by using organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD), which leads to selective injury of the CA1 subregion 24 h later. We developed models of pharmacological preconditioning by exposing slices to subtoxic concentrations of either N-methyl-D-aspartate (NMDA) or (S)-3,5-dihydroxyphenylglycine (DHPG) and then, 24 h later, to 30 min OGD. Under these conditions, we observed a significant reduction in OGD-induced CA1 damage. Exposure of slices to the PARP-1 and -2 inhibitors TIQ-A, PJ-34 and UPF 1069 during preconditioning prevented the development of OGD tolerance in a concentration-dependent manner. NMDA and DHPG preconditioning increased the activity of PARP, as detected by immunoblots using antibodies against the poly(ADP-ribose) polymer product, but was not associated with consumption of cellular NAD(+) or ATP. Neuroprotection induced by preconditioning was also prevented by the caspase inhibitor Z-VAD-FMK. The modest but significant increase in caspase-3/7 induced by preconditioning, however, was not associated with PARP-1 cleavage, as occurred with staurosporine. Finally, TIQ-A prevented the activation of ERK1/2 and Akt induced by NMDA preconditioning, suggesting that the protective mechanism evoked by PARP requires activation of these prosurvival mediators. Our results suggest that preconditioning with appropriate pharmacological stimuli may promote neuroprotective mechanisms triggered by the sublethal activation of two otherwise deleterious executioners such as PARP and caspase-3/7.

Rat hippocampal slice culture models for the evaluation of neuroprotective agents.

Organotypic slices cultured for weeks in vitro represent an extremely valuable strategy for the investigation of the long-term properties of neuronal circuits under physiological and pathological conditions. Here, we describe how to prepare rat organotypic hippocampal slice cultures and how to expose them for appropriate periods of time to excitotoxic agents or to oxygen and glucose deprivation conditions, in order to mimic the pattern of pyramidal cell damage which is observed in vivo and in other in vitro models. This preparation is very useful not only to study synaptic plasticity or the pathways and mechanisms of neurodegeneration but also to evaluate the effects of neuroprotective agents.

In vitro comparison of the cytotoxic effects of clinically available ophthalmic solutions of fluoroquinolones on human keratocytes.

OBJECTIVE: To compare the cytotoxic effects of preserved versus unpreserved commercially available ophthalmic preparations of fluoroquinolones on human keratocytes in vitro. DESIGN: Experimental study. METHODS: Human keratocytes in vitro were incubated for 15 or 60 minutes with commercially available preparations containing different types of fluoroquinolones, with or without benzalkonium chloride. We examined the morphologic aspects of the cultures by an inverted-phase contrast microscope and the release of cytoplasmic enzyme lactate dehydrogenase into the medium immediately or 24 hours after exposure to drugs. RESULTS: Whereas preparations of ofloxacin, norfloxacin, and gatifloxacin, all containing benzalkonium chloride, and moxifloxacin, which is preservative-free, displayed various degrees of cytotoxicity in our model, the unpreserved monodose preparation of norfloxacin was virtually devoid of harmful effects under our experimental conditions. CONCLUSIONS: Our in vitro results indicated the cytotoxic role of preservatives in commercial preparations of fluoroquinolones and the relative nontoxicity of monodose unpreserved norfloxacin, even when keratocytes were incubated with this formulation for 6 hours.

CB1 receptors and post-ischemic brain damage: studies on the toxic and neuroprotective effects of cannabinoids in rat organotypic hippocampal slices.

Cannabinoids (CBs) are implicated in a number of physiological and pathological mechanisms in the central nervous system, but their exact role in post-ischemic brain injury is unclear. The toxic and neuroprotective effects of synthetic and endogenous CBs were evaluated in rat organotypic hippocampal slices exposed to 20 min oxygen-glucose deprivation (OGD) and in gerbils subjected to bilateral carotid occlusion for 5 min. When present in the incubation medium, the synthetic CB agonists WIN 55212-2 and CP 55940 (1-30 µM) and the CB1 agonist ACEA exacerbated CA1 injury induced by OGD, whereas the CB1 receptor antagonists AM 251 and LY 320135 were neuroprotective with maximal activity at 1 µM. AM 251 (at 3 mg/kg, i.p.) also attenuated CA1 pyramidal cell death in gerbils in vivo. The endocannabinoid 2-arachidonoylglycerol (2-AG) reduced OGD injury in hippocampal slices at 0.1-1 µM, whereas anandamide (AEA) was neurotoxic at the same concentrations. The effects of WIN 55212-2, AEA and 2-AG in slices were all dependent on the activation of CB1 but not CB2 receptors, except for the toxic effects of AEA that were also dependent on vanilloid TRPV1 receptors. Our results suggest that exogenous administration of CB1 agonists and the production of endocannabinoids "on demand" may produce different, if not opposite, effects on the fate of neurons following cerebral ischemia.

Involvement of endocannabinoid signaling in the neuroprotective effects of subtype 1 metabotropic glutamate receptor antagonists in models of cerebral ischemia.

Experimental evidence indicates that metabotropic glutamate (mGlu) receptors of the mGlu1 and mGlu5 subtypes play a differential role in models of cerebral ischemia and that only mGlu1 receptors are implicated in the pathways leading to postischemic neuronal injury. The localization of mGlu1 receptors in GABA-containing interneurons rather than in hippocampal CA1 pyramidal cells that are vulnerable to ischemia has prompted experimental studies that have demonstrated mGlu1 receptor antagonist agents attenuate postischemic injury by enhancing GABA-mediated neurotransmission, thus providing a new viewpoint on the neuroprotective mechanism of these pharmacological agents. In view of the recent discovery of a functional interaction between group I mGlu receptors and the cannabinoid system in the modulation of synaptic transmission, we propose a novel mechanism that predicts that the neuroprotective effects of mGlu1 receptor antagonists on CA1 pyramidal cells are mediated by a mechanism that overcomes the "synaptic circuit break" operated by endocannabinoids on GABAergic transmission.

Activation of the histaminergic H3 receptor induces phosphorylation of the Akt/GSK-3 beta pathway in cultured cortical neurons and protects against neurotoxic insults.

Stimulation of histamine H(3) receptors (H(3)R) activates G(i/o)-proteins that inhibit adenylyl cyclase and triggers MAPK and phospholipase A(2). In a previous study, we showed that H(3)R-mediated phosphorylation of Akt at Ser473 occurs in primary cultures of rat cortical neurons, but neither the downstream targets nor the function of such activation were explored. In this report we address these questions. Western blotting experiments showed that H(3)R-mediated activation of Akt in cultured rat cortical neurons was inhibited by LY 294004 and U0126, suggesting that it depends on phosphoinositide-3-kinase and mitogen-activated protein kinase kinase. H(3)R activation phosphorylated, hence inactivated, the Akt downstream effector glycogen synthase kinase-3beta, increased the expression of the antiapoptotic protein Bcl-2 and protected cultured rat and mouse cortical neurons from neurotoxic insults in a dose-dependent manner. All these effects were inhibited by the H(3)R antagonist inverse/agonist thioperamide. Mouse cortical cells expressed H(3)R as revealed by immunostaining experiments, and stimulation of H(3)R phoshorylated Akt and decreased caspase 3 activity. Hence, we uncovered a yet unexplored action of the H(3)R that may help understand the impact of H(3)R signaling in the CNS.

Neuroprotection by group I mGlu receptors in a rat hippocampal slice model of cerebral ischemia is associated with the PI3K-Akt signaling pathway: a novel postconditioning strategy?

Ischemic postconditioning is defined as a repetitive series of brief interruptions of reperfusion applied immediately after ischemia. In this study, postconditioning was investigated by first exposing rat organotypic hippocampal slices to 30min oxygen-glucose deprivation (OGD), which promotes selective CA1 pyramidal cell death, and 5min later to either a brief period (3min) of OGD or to a low dose (10microM) of 3,5-dihydroxyphenylglycine (DHPG) for 30min. Both protocols attenuated CA1 neuronal injury, as revealed 24h later by measuring the intensity of propidium iodide fluorescence in this region. The beneficial effects were observed when DHPG postconditioning was applied up to 15min after OGD, but not at later time points, and was not additive with the neuroprotective effects of a preconditioning DHPG treatment. The attenuation of the OGD-induced CA1 injury evoked by postconditioning was prevented when mGlu1 and mGlu5 receptor antagonists and inhibitors of phosphatidylinositol 3-kinase and Akt activity were present in the incubation medium during the 5min recovery period after OGD and the 30min exposure to DHPG. The PI3K inhibitor was also able to prevent the reduction of NMDA toxicity induced by the DHPG treatment. Finally, DHPG increased the phosphorylation of Akt in a transient and mGlu1/mGlu5-dependent manner. Our results show that activation of the mGlu1/mGlu5-PI3K-Akt signaling pathway plays a crucial role in the mechanisms of postconditioning evoked by DHPG and point to this strategy as a possible novel therapeutic tool for stroke and cerebral ischemia.

Differential role of mGlu1 and mGlu5 receptors in rat hippocampal slice models of ischemic tolerance.

Activation of glutamate receptors has been proposed as a key factor in the induction of ischemic tolerance. We used organotypic rat hippocampal slices exposed to 30 min oxygen-glucose deprivation (OGD) to evaluate postischemic pyramidal cell death in the CA1 subregion. In this model, 10 min exposure to OGD 24 h before the exposure to toxic OGD was not lethal and reduced the subsequent OGD neurotoxicity by approximately 53% (ischemic preconditioning). Similarly, a 30 min exposure to the group I mGlu receptor agonist DHPG (10 microM) significantly reduced OGD neurotoxicity 24 h later (pharmacological preconditioning). Ischemic tolerance did not develop when either the selective mGlu1 antagonists LY367385 and 3-MATIDA or the AMPA/KA antagonist CNQX were present in the incubation medium during exposure to sublethal OGD. Neither the NMDA antagonist MK801 nor the mGlu5 antagonist MPEP affected the preconditioning process. On the other hand, pharmacological preconditioning was prevented not only by LY367385 or CNQX, but also by MPEP. In preconditioned slices, the toxic responses to AMPA or NMDA were reduced. The neurotoxicty of 100 microM DHPG in slices simultaneously exposed to a mild (20 min) OGD was differentially altered in the two preconditioning paradigms. After ischemic preconditioning, DHPG neurotoxicity was reduced in a manner that was sensitive to LY367385 but not to MPEP, whereas after pharmacological preconditioning it was enhanced in a manner that was sensitive to MPEP but not to LY367385. Our results show that mGlu1 and mGlu5 receptors are differentially involved in the induction and expression of ischemic tolerance following two diverse preconditioning stimuli.