Neuroprotective effects of arachidonic acid against oxidative stress on rat hippocampal slices

Arachidonic acid (AA), 5,8,11,14-eicosateraenoic acid is abundant, active and necessary in the human body. In the present study, we reported the neuroprotective effects and mechanism of arachidonic acid on hippocampal slices insulted by glutamate, NaN(3) or H(2)O(2)in vitro. Different types of models of brain injury in vitro were developed by 1mM glutamate, 10mM NaN(3) or 2mM H(2)O(2). After 30 min of preincubation with arachidonic acid or linoleic acid, hippocampal slices were subjected to glutamate, NaN(3) or H(2)O(2), then the tissue activities were evaluated by using the 2,3,5-triphenyltetrazolium chloride method. Endogenous antioxidant enzymes activities (SOD, GSH-PX and catalase) in hippocampal slices were evaluated during the course of incubation. MK886 (5 microM; a noncompetitive inhibitor of proliferator-activated receptor [PPAR]alpha), BADGE (bisphenol A diglycidyl ether; 100 microM; an antagonist of PPARgamma) and cycloheximide (CHX; 30 microM; an inhibitor of protein synthesis) were tested for their effects on the neuroprotection afforded by arachidonic acid. Population spikes were recorded in randomly selected hippocapal slices. Arachidonic acid (1-10 microM) dose dependently protected hippocampal slices from glutamate and H(2)O(2) injury (P<0.01), and arachidonic acid (10 microM) can significantly improve the activities of Cu/Zn-SOD in hippocampal slices after 1h incubation. In addition, 10 microM arachidonic acid significantly increased the activity of Mn-SOD and catalase, and decreased the activities of Cu/Zn-SOD to control value after 3h incubation. These secondary changes of SOD during incubation can be reversed by indomethacine (10 microM; a nonspecific cyclooxygenase inhibitor) or AA 861 (20 microM; a 5-lipoxygenase inhibitor). Its neuroprotective effect was completely abolished by BADGE and CHX. These observations reveal that arachidonic acid can defense against oxidative stress by boosting the internal antioxidant system of hippocampal slices. Its neuroprotective effect may be mainly mediated by the activation of PPARgamma and synthesis of new protein in tissue.     

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.     

Redox Modulation of N-Methyl-D-Aspartate-Stimulated Neurotransmitter Release from Rat Brain Slices

Rat brain cortical slices released tritiated norepinephrine ([3H]NA) during a 2-min stimulation with N-methyl-D-aspartate (NMDA). Dithiothreitol (DTT; 0.1-5 mM), present for 6 min prior to stimulation, dose-dependently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 microM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose-response curve of NMDA to the left. Cortical slices released approximately the same amount of [3H]NA with 10 microM NMDA following DTT treatment (about 5%) as non-DTT-treated control slices did with 500 microM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA-stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7-chlorokynurenic acid, and the competitive NMDA antagonist, 2-aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA-stimulated responses.     

S-Allyl-L-cysteine attenuates cerebral ischemic injury by scavenging peroxynitrite and inhibiting the activity of extracellular signal-regulated kinase

S-Allyl-L-cysteine (SAC) has been shown to reduce ischemic injury due to its antioxidant activity. However, the antioxidant property of SAC has been controversial. The present study investigated the neuroprotective mechanism of SAC in cerebral ischemic insults. SAC decreased the size of infarction after transient or global ischemic insults. While it did not alter the N-methyl-D-aspartate excitotoxicity, SAC significantly scavenged the endogenously or exogenously produced ONOO- and reduced ONOO- cytotoxicity. In contrast, SAC has much lower scavenging activity against H2O2, O2*(-) or NO. Further, SAC inhibited the activity of extracellular signal-regulated kinase (ERK) increased in cultured neurons exposed to oxygen-glucose deprivation or in rat brain tissue after transient middle cerebral artery occlusion. The neuroprotective effect of SAC was mimicked by the ERK inhibitor U0125. The present results indicate that SAC exert its neuroprotective effect by scavenging ONOO- and inhibiting the ERK signaling pathway activated during initial hypoxic/ischemic insults.     

Mechanism of guanosine-induced neuroprotection in rat hippocampal slices submitted to oxygen-glucose deprivation

Guanine derivates have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. Guanine derivatives are spontaneously released to the extracellular space from cultured astrocytes during oxygen-glucose deprivation (OGD) and may act as trophic factors, glutamate receptors blockers or glutamate transport modulators, thus promoting neuroprotection. The aim of this study was to evaluate the mechanisms involved in the neuroprotective role of the nucleoside guanosine in rat hippocampal slices submitted to OGD, identifying a putative extracellular binding site and the intracellular signaling pathways related to guanosine-induced neuroprotection. Cell damage to hippocampal slices submitted to 15 min of OGD followed by 2 h of reperfusion was decreased by the addition of guanosine (100 microM) or guanosine-5'-monophosphate (GMP, 100 microM). The neuroprotective effect of guanosine was not altered by the addition of adenosine receptor antagonists, nucleosides transport inhibitor, glutamate receptor antagonists, glutamate transport inhibitors, and a non-selective Na(+) and Ca(2+) channel blocker. However, in a Ca(2+)-free medium (by adding EGTA), guanosine was ineffective. Nifedipine (a Ca(2+) channel blocker) increased the neuroprotective effect of guanosine and 4-aminopyridine, a K(+) channel blocker, reversed the neuroprotective effect of guanosine. Evaluation of the intracellular signaling pathways associated with guanosine-induced neuroprotection showed the involvement of PKA, PKC, MEK and PI-3 K pathways, but not CaMKII. Therefore, this study shows guanosine is acting via K(+) channels activation, depending on extracellular Ca(2+) levels and via modulation of the PKA, PKC, MEK and/or PI-3 K pathways.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 M) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.     

Protection by cholesterol-extracting cyclodextrins: a role for N-methyl-D-aspartate receptor redistribution

Cyclodextrins (CDs) are cyclic oligosaccharides composed of a lipophilic central cavity and a hydrophilic outer surface. Some CDs are capable of extracting cholesterol from cell membranes and can affect function of receptors and proteins localized in cholesterol-rich membrane domains. In this report, we demonstrate the neuroprotective activity of some CD derivatives against oxygen-glucose deprivation (OGD), N-methyl-D-aspartic acid (NMDA) and glutamate in cortical neuronal cultures. Although all CDs complexed with NMDA or glutamate, only beta-, methylated beta- and sulfated beta-CDs displayed neuroprotective activity and lowered cellular cholesterol. Only CDs that lowered cholesterol levels redistributed the NMDA receptor NR2B subunit, PSD-95 (postsynaptic density protein 95 kDa) and neuronal nitric oxide synthase (nNOS) from Triton X-100 insoluble membrane domains to soluble fractions. Cholesterol repletion counteracted the ability of methylated beta-CD to protect against NMDA toxicity, and reversed NR2B, PSD-95 and nNOS localization to Triton X-100 insoluble membrane fraction. Surprisingly, neuroprotective CDs had minimal effect on NMDA receptor-mediated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), but did suppress OGD-induced increases in [Ca(2+)](i). beta-CD, but not Mbeta-CD, also caused a slight block of NMDA-induced currents, suggesting a minor contribution to neuroprotection by direct action on NMDA receptors. Taken together, data suggest that cholesterol extraction from detergent-resistant microdomains affects NMDA receptor subunit distribution and signal propagation, resulting in neuroprotection of cortical neuronal cultures against ischemic and excitotoxic insults. Since cholesterol-rich membrane domains exist in neuronal postsynaptic densities, these results imply that synaptic NMDA receptor subpopulations underlie excitotoxicity, which can be targeted by CDs without affecting overall neuronal Ca(2+) levels.     

Conditioning Heat Stress Reduces Excitotoxic and Apoptotic Components of Oxygen-Glucose Deprivation-Induced Neuronal Death In Vitro

Abstract: We investigated the effects of sublethal heat stress in murine cortical cell cultures exposed to combined oxygen and glucose deprivation. Pretreatment with sublethal heat stress mildly attenuated the widespread neuronal death induced a day later by 30–60 min of oxygen-glucose deprivation. Heat stress also blunted the increase in extracellular glutamate concentrations induced by the oxygen-glucose deprivation, as well as the neuronal death and ⁴⁵Ca²⁺ uptake induced by exogenous addition of NMDA, although no reduction was seen in neuronal death caused by exogenous kainate or in NMDA-induced whole-cell currents. However, arguing against the idea that the neuroprotective effect of heat stress against neuronal death was exclusively due to reduction of excitotoxicity was the finding that heat stress also reduced the neuronal apoptosis induced by oxygen-glucose deprivation in the presence of glutamate antagonists. This antiapoptotic effect was specific in that heat stress did not reduce neuronal vulnerability to staurosporine-induced apoptosis. Whereas heat stress transiently suppressed protein synthesis, achieving comparable protein synthesis inhibition with cycloheximide did not reproduce the neuroprotective effects of heat stress. These studies suggest that a conditioning heat stress is able to attenuate both the excitotoxic and the apoptotic components of oxygen-glucose deprivation-induced neuronal death in vitro, by mechanisms independent of protein synthesis reduction.     

[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors

[3H]Norepinephrine ([3H]NE) efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) greater than kainic acid (KA) greater than L-glutamate greater than or equal to D,L-homocysteate greater than L-aspartate greater than quinolinic acid greater than quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50 = 69 microM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonohepatanoic acid. gamma-D-Glutamylglycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 microM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.     

NAP protects hippocampal neurons against multiple toxins

The femtomolar-acting protective peptide NAP (NAPVSIPQ), derived from activity-dependent neuroprotective protein (ADNP), is broadly neuroprotective in vivo and in vitro in cerebral cortical cultures and a variety of cell lines. In the present study, we have extended previous results and examined the protective potential of NAP in primary rat hippocampal cultures, using microtubule-associated protein 2 (MAP2) as a measure for neuroprotection. Results showed that NAP, at femtomolar concentrations, completely protected against oxygen-glucose deprivation, and cyanide poisoning. Furthermore, NAP partially protected against kainic acid excitotoxicity. In summary, we have significantly expanded previous findings in demonstrating here direct neuroprotective effects for NAP on vital hippocampal neurons that are key participants in cognitive function in vivo.     

NMDA-induced neuroprotection in hippocampal neurons is mediated through the protein kinase A and CREB (cAMP-response element-binding protein) pathway

N-Methyl-d-aspartate (NMDA) receptors play a critical role in the brain stimulating synaptic plasticity and mediating neurodegeneration; a neuroprotective role has also been described, but its molecular mechanisms in hippocampus are under study. Here, we report that in primary cultures of rat hippocampal neurons exposure to low micromolar NMDA concentrations are neuroprotective against excitotoxic insults, while high micromolar NMDA concentrations provoke neuronal death. Molecular analysis reveals that a toxic concentration of NMDA induced a transient phosphorylation of cAMP-response element-binding protein (pCREB) in 2 min that rapidly decreased below basal levels. In contrast, a nontoxic NMDA concentration gave up to longer (20 min) rise of pCREB, suggesting that neuroprotection could be associated to a relatively prolonged presence of pCREB in the neurons. In support of this tenet, rolipram, an inhibitor of phosphodiesterase IV that increases the levels of cAMP and pCREB, protected against NMDA-induced neuronal death. Similar results were obtained with dibutyrate-cAMP (a cAMP analogue with membrane permeability) that also abrogated NMDA excitotoxicity. Conversely, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H89), an inhibitor of protein kinase A (PKA), that prevents the formation of pCREB induced by nontoxic NMDA concentrations, reverted the neuroprotection achieved by preincubation of low micromolar NMDA concentrations. These results substantiate the notion that induction of pCREB via PKA plays an important role in NMDA-mediated neuroprotection.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Kynurenine and glycine enhance neuronal sensitivity to N-methyl-D-aspartate

Neurones in rat hippocampal slices were excited by microiontophoretic applications of N-methyl-D-aspartate (NMDA) and kainate. Responses to NMDA were potentiated by glycine 300 microM or 1 mM in the perfusing medium. A small potentiation of kainate was not observed in the presence of the NMDA antagonist 2-amino-5-phosphonopentanoic acid (2AP5). The potentiation of NMDA responses by glycine was not prevented by strychnine 5 or 30 microM and was also shown by D-serine and L-kynurenine but not L-leucine. If sensitivity to NMDA was reduced by kynurenic acid, glycine and L-kynurenine produced a greater enhancement of NMDA. The requirement of NMDA receptor activation for the occupation of strychnine-resistant glycine sites can thus be demonstrated in complex systems such as brain slices. It is possible that L-kynurenine may also be an endogenous ligand capable of modulating NMDA sensitivity.     

Biochemical Responses Mediated by N-Methyl-d-Aspartate Receptors in Rat Cortical Slices Are Differentially Sensitive to Magnesium

The effects of magnesium on the inhibition of phosphoinositide (PI) hydrolysis and the stimulation of [3H]norepinephrine release by N-methyl-D-aspartate (NMDA) in rat cortical slices were investigated. Removal of the magnesium from the buffer resulted in a small reduction of the inhibitory effect of 100 microM NMDA (34% inhibition in the absence of magnesium, compared with 51% for the control) when slices were coincubated with NMDA and carbachol. Addition of 10 mM Mg2+ also allowed the inhibitory effect of 100 microM NMDA on carbachol-stimulated PI hydrolysis to be expressed (44% inhibition) under these conditions. Concentration-effect curve analysis for the NMDA-induced inhibition of carbachol-stimulated PI hydrolysis indicated that the IC50 for NMDA was decreased from 14.9 microM for the control to 4.2 microM in the absence of magnesium. The absence of magnesium also had small effects on the concentration-effect curve for (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate reversal of the inhibitory effects of NMDA on carbachol-stimulated PI hydrolysis. The absence of magnesium also shifted slightly downward and flattened the NMDA concentration-effect curve if the cortical slices were pretreated with NMDA in the presence or absence of magnesium followed by removal of the NMDA and subsequent stimulation with carbachol. In contrast, cortical slices that had been prepared and treated similarly to the slices used in the PI experiments were very sensitive to the inhibitory effects of magnesium when using the NMDA stimulation of [3H]norepinephrine release assay in the presence or absence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroprotective actions of deferiprone in cultured cortical neurones and SHSY-5Y cells

Alzheimer's disease (AD) is a common neurodegenerative disorder, but the initiating molecular processes contributing to neuronal death are not well understood. AD is associated with elevated soluble and aggregated forms of amyloid beta (Abeta) and with oxidative stress. Furthermore, there is increasing evidence for a detrimental role of iron in the pathogenic process. In this context, iron chelation by compounds such as 3-hydroxypyridin-4-one, deferiprone (Ferriprox) may have potential neuroprotective effects. We have evaluated the possible neuroprotective actions of deferiprone against a range of AD-relevant insults including ferric iron, H(2)O(2) and Abeta in primary mouse cortical neurones. We have investigated the possible neuroprotective actions of deferiprone (1, 3, 10, 30 or 100 microM) in primary neuronal cultures following exposure to ferric iron [ferric nitrilotriacetate (FeNTA); 3 and 10 microM], H(2)O(2) (100 microM) or Abeta1-40 (3, 10 and 20 microM). Cultures were treated with deferiprone or vehicle either immediately or up to 6 h after the insult in a 24-well plate format. In order to elucidate a possible neuroprotective action of deferiprone against Parkinson's disease relevant insults another group of experiments were performed in the human neuroblastoma catecholaminergic SHSY-5Y cell line. SHSY-5Y cells were treated with MPP(+) iodide, the active metabolite of the dopaminergic neurotoxin MPTP and the neuroprotective actions of deferiprone evaluated. Cytotoxicity was assessed at 24 h by lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide turnover (FeNTA and hydrogen peroxide) and morphometric analysis of cell viability by Hoechst 33324/propidium iodide (FeNTA, Abeta and MPP(+)) or 6-carboxyfluorescein diacetate and annexin V-Cy3 (Abeta). The present study demonstrates that deferiprone protects against FeNTA, hydrogen peroxide, MPP(+) and Abeta1-40-induced neuronal cell death in vitro, which is consistent with previous in vitro and in vivo studies that have demonstrated similar protection with other iron chelators.     

N-methyl-D-aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV-1 envelope protein gp120, external pH and protein kinase C inhibition

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [(3)H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 microM or 1 mM) plus glycine (1 microM)-elicited [(3)H]NA, but not [(3)H]DA release. The NMDA (100 microM)-evoked release of [(3)H]NA and [(3)H]DA was similar and concentration-dependently enhanced by glycine or D-serine (0.1-1 microM); in contrast, the HIV-1 envelope protein gp120 potently (30-100 pM) enhanced the NMDA-evoked release of [(3)H]NA, but not that of [(3)H]DA. Gp120 also potentiated quinolinate-evoked [(3)H]NA release. Ifenprodil (0.1-0.5 microM) or CP-101,606 (0.1-10 microM) inhibited the NMDA plus glycine-evoked release of both [(3)H]catecholamines. Zinc (0.1-1 microM) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [(3)H]NA elicited by NMDA plus glycine, whereas the release of [(3)H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 microM) or H7 (10 microM) selectively prevented the effect of NMDA plus glycine on the release of [(3)H]NA. GF 109203X also blocked the release of [(3)H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.     

A role for the protein phosphatase 2B in altered hippocampal synaptic plasticity in the aged rat.

Synaptic plasticity following NMDA application on hippocampal slices from young (3-5 months) and aged (24-27 months) rats was compared. In young rats, NMDA (20 microM) induced opposite effects depending on the duration of the application. A short (1 min) or long (5 min) application induced a long-term depression of synaptic activity while a 3 min application induced a potentiation. In aged rats, however, NMDA application always induced depression, regardless of the duration. To identify mechanisms which could explain the difference observed between young and aged rats, we explored changes in NMDA receptor activation and changes in kinase/phosphatase balance. We first demonstrate that the potentiation present in slices from young rats was not restored in aged rats by exogenous application of the co-agonist of NMDA receptor d-serine (which compensates for the changes in NMDAR activation seen in aged rats). This suggested that alterations in synaptic plasticity activation mainly involve intracellular mechanisms. We next showed that the participation of the kinases PKA and CaMKII in the NMDA-induced potentiation in young rats is negligible. Finally, we determined the consequences of phosphatase inhibition in aged rats. Incubation of slices in okadaic acid (a PP1/PP2B antagonist) did not affect the depression induced by a 3min NMDA application in aged rats. The PP2B antagonist FK506 restored potentiation in aged rats (3 min NMDA application). In hippocampal neurons from aged rats, a depression is always observed, suggesting a preferential activation of PP2B by NMDA in these neurons.     

Effects of the mono- and tetrasialogangliosides GM1 and GQ1b on ATP-induced long-term potentiation in hippocampal CA1 neurons

The effects of the mono- and tetrasialogangliosides, GM1 and GQ1b, on ATP-induced long-term potentiation (LTP) were studied in CA1 neurons of guinea pig hippocampal slices. Application of 5 or 10 microM ATP for 10 min resulted in a transient depression followed by a slow augmentation of synaptic transmission, leading to LTP. LTP induced by treatment with 5 microM ATP was facilitated in hippocampal slices prepared from animals treated for 6 days with a ceramide analog, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propranol, which stimulates ganglioside biosynthesis. In addition, LTP induced by 5 microM ATP was significantly enhanced when naive slices were incubated with GQ1b but not with GM1. These results suggest that a cooperative effect between extracellular ATP and GQ1b enhances ATP-induced LTP in hippocampal CA1 neurons. In addition, the LTP induced by 10 microM ATP was blocked by coapplication of the NMDA antagonist AP5 (5 microM or 50 microM), and this effect was partially inhibited by GQ1b pretreatment of the slices, suggesting that in hippocampal CA1 neurons, the enhancing effect of GQ1b on ATP-induced LTP is mediated by modulation of NMDA receptors/Ca(2+) channels.     

Functional identification of neuroprotective molecules

The central nervous system has the capacity to activate profound neuroprotection following sub-lethal stress in a process termed preconditioning. To gain insight into this potent survival response we developed a functional cloning strategy that identified 31 putative neuroprotective genes of which 28 were confirmed to provide protection against oxygen-glucose deprivation (OGD) or excitotoxic exposure to N-methyl-D-aspartate (NMDA) in primary rat cortical neurons. These results reveal that the brain possesses a wide and diverse repertoire of neuroprotective genes. Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.