BDNF prevents NO mediated glutamate cytotoxicity in cultured cortical neurons

The effects of brain-derived neurotrophic factor (BDNF) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. BDNF induced TrkB tyrosine phosphorylation in rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate and incubated with normal medium for 24 h. Glutamate cytotoxicity was prevented by MK-801, which is a non-competitive blocker of N-methyl-D-aspartate and N(omega)-nitro-L-arginine, which is a blocker of nitric oxide synthetase. Delayed neurotoxicity was also induced by ionomycin, a calcium ionophore, and nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1). Incubating cultures with BDNF for 10 min to 24 h protected cortical neurons against glutamate neurotoxicity. The protective effects of BDNF against glutamate cytotoxicity were dependent on both its concentrations and incubation time. BDNF also prevented the ionomycin-, SNOC-, and SIN-1 induced cytotoxicity. These results indicate that BDNF protects cultured cortical neurons from NMDA receptor-mediated glutamate neurotoxicity by reducing cytotoxic action of NO.     

Ether extract of fetal calf serum protects cultured rat cortical neurons against glutamate cytotoxicity

OBJECTIVE: To elucidate the role of Helicobacter pylori in relapsing disease after partial gastrectomy for peptic ulcer. DESIGN: Retrospective study of gastroscopies between January 1985 and February 1988. SETTING: Department of Surgery, Helsinki University Central Hospital, Finland. PARTICIPANTS: One hundred and fifty-five patients, who had undergone partial gastrectomy for peptic ulcer disease. MAIN OUTCOME MEASURES: Correlation between clinical and laboratory data, macroscopic findings at gastroscopy and histopathology. RESULTS: At gastroscopy 41 patients showed an ulcer at the site of anastomosis or in the gastric stump and two patients had a history of a previous ulcer recurrence. The median time interval between operation and relapse was 4 years. There was no correlation between ulcer recurrence, sex, age, ABO blood group or other diseases. Smokers and patients using non-steroidal anti-inflammatory drugs (NSAIDs) or alcohol had more relapses, but the difference was not significant. The recurrence rate was higher after Billroth II (BII; 34%) than after Roux-en-Y (14%; P = 0.03) or Billroth I (BI) reconstruction (24%). Giemsa staining demonstrated H. pylori in the gastric stump of 37% of the patients. H. pylori expression was related to age but unrelated to sex, ABO blood group, NSAID use, smoking or alcohol consumption. H. pylori positivity was more common (52%) after BI than after BII (28%; P = 0.04) or Roux-en-Y resection (40%). Recurrent ulcer was more often found in gastric remnants with normal mucosa (36%) than in those with H. pylori-positive gastritis (18%; P = 0.03) or H. pylori-negative gastritis (26%). CONCLUSION: It seems that H. pylori infection plays a minor role in the pathogenesis of ulcer recurrence after partial gastrectomy for peptic ulcer disease. Eradication of H. pylori of the remnant stomach is therefore presumably not effective in preventing ulcer recurrence.     

Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones

Domoic acid, a naturally occurring kainoid, has been responsible for several outbreaks of fatal poisoning after shellfish ingestion, and we examined its neurotoxic mechanism in cultured murine cortical neurones. Using observations of neuronal viability and morphology, exposure to domoic acid for 24 h was found to induce substantial concentration-dependent neuronal cell death. Domoic acid-mediated neuronal death was attenuated by the non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-selective antagonist LY293558 ((3S,4aR,6R,8aR)-6-[2-(1H-tetrazol-5-yl)-ethyl]-1,2,3, 4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid), but unaffected by NS-102 (5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2, 3-dione-3-oxime)--a low-affinity kainate receptor antagonist. Domoic acid was equipotent with (S)-AMPA (EC50 values 3.8 and 3.4 microM respectively); however, (S)-AMPA induced only 50% cell death compared to > 80% cell death induced by domoic acid. Kainate also killed > 80% of cortical neurones; however, domoic acid was about 19 times more potent than kainate (EC50 75 microM). We show the potent neurotoxicity of domoic acid for the first time in a pure neuronal model and indicate that domoic acid acts via high-affinity AMPA- and kainate-sensitive glutamate receptors to produce excitotoxic cell death.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

Rapid, activation-induced redistribution of ionotropic glutamate receptors in cultured hippocampal neurons

We have examined the membrane localization of an AMPA receptor subunit (GluR1) and an NMDA receptor subunit (NR1) endogenously expressed in primary cultures of rat hippocampal neurons. In unstimulated cultures, both GluR1 and NR1 subunits were concentrated in SV2-positive synaptic clusters associated with dendritic shafts and spines. Within 5 min after the addition of 100 microM glutamate to the culture medium, a rapid and selective redistribution of GluR1 subunits away from a subset of synaptic sites was observed. This redistribution of GluR1 subunits was also induced by AMPA, did not require NMDA receptor activation, did not result from ligand-induced neurotoxicity, and was reversible after the removal of agonist. The activation-induced redistribution of GluR1 subunits was associated with a pronounced (approximately 50%) decrease in the frequency of miniature EPSCs, consistent with a role of GluR1 subunit redistribution in mediating rapid regulation of synaptic efficacy. We conclude that ionotropic glutamate receptors are regulated in native neurons by rapid, subtype-specific membrane trafficking, which may modulate synaptic transmission in response to physiological or pathophysiological activation.     

Ketamine and nifedipine protect cultured cortical neurons against injurious effect of glutamate]

The effects of glutamate on cultured cortical neurons and the protective effect of ketamine and nifedipine were studied. On day 10 after plating of the cortical cells from 16-18 day-old fetal rats, the cultures were exposed to 50 mumol.L-1 glutamate and low glucose (1 g.L-1) for 10 min-24 h. The results showed that a release of lactate dehydrogenase (LDH) into the culture supernatant was observed as a function of time. The values of LDH efflux in culture medium was significantly lower than those of controls when the cells were pretreated with ketamine or nifedipine 10 min prior to addition of glutamate. More significant decrease of LDH activity in culture medium was observed when the two drugs were used in combination. These results demonstrate that the dissociated cultured cortical neurons from fetal rat are seriously damaged by glutamate. Such damage could be attenuated by ketamine and nifedipine, suggesting that ketamine and nifedipine may protect neurons from the glutamate toxicity, and the effect of combining ketamine and nifedipine was greater than either ketamine or nifedipine alone.     

Ifenprodil discriminates subtypes of the N-methyl-D-aspartate receptor: selectivity and mechanisms at recombinant heteromeric receptors

Each of 28 nonautistic children with attention-deficit hyperactivity disorder and mental retardation received placebo, methylphenidate (0.4 mg/kg/day), and fenfluramine (gradually increased to 1.5 mg/kg/day) for 4 weeks each in a double-blind, crossover design. Teacher ratings indicated significant improvements with both active drugs on subscales designated as Conduct Problem, Hyperactivity, and Irritability, but methylphenidate alone produced improvements on an Inattention subscale. Parent ratings indicated significant improvements with both drugs on subscales labeled Hyperactivity, Motor Excess, and Conduct Problem. Fenfluramine alone caused improved parent ratings on Irritability and Inappropriate Speech, and on Conners' Abbreviated Symptom Questionnaire. Unlike a previous study, subgroup analyses failed to show a significantly better clinical response to methylphenidate for subjects with higher mental ages, although children with higher IQs responded better than those with IQs less than 45. The active drugs had contrasting effects on heart rate and blood pressure. Fenfluramine caused significant weight reductions relative to both placebo and methylphenidate. These findings suggest that both methylphenidate and fenfluramine have useful, but somewhat different, clinical effects in certain children with attention-deficit hyperactivity disorder and mental retardation.     

Ethanol prevents the glutamate release induced by N-methyl-D-aspartate in the rat striatum

The administration of ethanol (2 g/kg, i.p.) or of the non-competitive antagonist(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloepten-5,1 0-imine maleate (MK-801; 1 mg/kg, i.p.) induced a decrease in the extracellular concentrations of glutamate, as studied by microdialysis in the striatum of awake rats. Moreover, ethanol and MK-801 completely prevented the increase in extraneuronal glutamate concentration induced by the focal application of N-methyl-D-aspartate (NMDA). The present results suggest that ethanol suppresses glutamate release through an inhibition of NMDA glutamate receptors in the rat striatum.     

Differential intracellular regulation of cortical GABA(A) and spinal glycine receptors in cultured neurons

The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.     

Diverse signal transduction pathways mediated by endogenous P2 receptors in cultured rat cerebral cortical neurons

The present study was conducted to assess the intracellular signaling pathways mediated by receptors for ATP, uridine triphosphate (UTP), and 2-methylthio ATP (2-MeSATP), by monitoring patch-clamp currents and intracellular calcium mobilization in cultured rat cortical cerebral neurons. All three agonists evoked potassium currents and increased the intracellular free Ca2+ concentration ([Ca2+]i), and these effects were inhibited by the broad G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) but not by the Gi/o-protein inhibitor pertussis toxin (PTX). UTP-evoked currents were inhibited by either the phospholipase C inhibitor neomycin or the selective protein kinase C (PKC) inhibitor GF109203X, and the rise in cytosolic Ca2+ was inhibited by either neomycin or the inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, indicating that the UTP receptor involved phospholipase C-mediated phosphatidylinositol signaling. In contrast, 2-MeSATP-induced currents and rise in cytosolic Ca2+ were not inhibited by either neomycin, or GF109203X, or heparin. 2-MeSATP elicited single-channel currents in the cell-attached patch-clamp configuration and also in excised patches. The G-protein activator GTP gamma S induced single-channel currents in a fashion that mimicked the effect of 2-MeSATP. These data suggest that 2 MeSATP activated potassium channels by a direct action of G-protein beta gamma subunits and increased [Ca2+]i by a mechanism independent of phospholipase C stimulation and IP3 production. ATP-evoked currents were partially inhibited by either neomycin or GF109203X, although the rise in cytosolic Ca2+ was not affected by these inhibitors. ATP produced single-channel currents with two major classes of the slope conductance (86 and 95 pS) in cell-attached patches, each of which is consistent with that achieved by 2-MeSATP (85 pS) or UTP (96 pS); the currents with the lower conductance were observed in the outside-out patch-clamp configuration. These results indicate that P2 receptors for UTP and 2-MeSATP are linked to a PTX-insensitive G-protein involving different signal transduction pathways and that ATP responses are mediated by both of these P2 receptors.     

Beta-amyloid prevents excitotoxicity via recruitment of glial glutamate transporters

Amyloid beta-protein (Abeta), a putative pathogenic endotoxin involved in Alzheimer's disease, induces redistribution of glutamate transporters in astrocytes and promotes their pump activity. Because the transporters are assumed to protect neurons against excitotoxicity by removing extracellular glutamate, we hypothesized that Abeta alters the vulnerability of neurons to glutamate. Cerebrocortical neuron-astroglial co-cultures were exposed to glutamate, the concentration of which was selected so that only 20% of the neurons exhibited degeneration. When cultures were pre-treated with Abeta, exposure to the same "mild" glutamate concentration failed to damage neurons. The Abeta-induced protection was abolished by a glial glutamate transporter inhibitor. Thus, Abeta can alleviate excitotoxicity through glutamate transporter activity. The present results may challenge prevailing concepts that Abeta-induced neuron loss causes Alzheimer's dementia and also provide practical insights into neuro-glial interactions in glutamate toxicity.     

Stimulatory effect of N-methyl-D-aspartate on somatostatin gene expression in cultured hypothalamic neurons

The aim of the present study was to determine whether N-methyl-D-aspartate (NMDA) stimulates somatostatin gene function in primary cultures of hypothalamic neurons. Neurons were either shortly (for 3, 8, 24 and 72 h) or chronically (for 11 days) exposed to NMDA (20 microM). Medium and cellular somatostatin contents were determined by radioimmunoassay, and steady-state preprosomatostatin mRNA levels by Northern blot analysis with an oligonucleotide probe. DNA content was measured as a cellular viability control. After 8 h incubation, NMDA induced a significant 2-fold increase in somatostatin mRNA accumulation, with a maximal 4-fold increase after 24 h incubation. A significant and dose-dependent (1.7-fold and 2.5-fold at 20 and 100 microM, respectively) stimulatory effect was also observed after chronic treatment. The kinetic patterns for medium and cellular somatostatin contents were similar to those obtained for somatostatin mRNA levels. Total DNA content was not modified under any experimental condition. The augmentations in cellular somatostatin and somatostatin mRNA determined after 24 h or chronic exposure to NMDA were blocked by (+)-5-methyl-10.11-dihydro-5H-dibenzo(a,d')cyclohepten-5,10-imine hydrogen maleate (MK-801), an NMDA receptor antagonist. MK-801 alone significantly (P < 0.05) reduced somatostatin mRNA. The stimulatory effect of NMDA on somatostatin mRNA was specific since it was not accompanied by any change in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. After immunostaining with a specific antibody against somatostatin, no difference was observed in the number of immunostained neurons detected in control and NMDA exposed groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual actions of nitric oxide in N-methyl-D-aspartate receptor-mediated neurotoxicity in cultured retinal neurons

This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) receptor-mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. The NOS activity measured by monitoring the conversion of [3H]arginine to [3H]citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. The concomitant addition of an inhibitor of NOS, Nomega-nitro-L-arginine (300 microM), with glutamate or NMDA reduced cell death by 70%. A brief exposure of the cells to sodium nitroprusside (SNP, 500 microM) or S-nitrosocysteine (SNOC, 500 microM), NO-generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA, or NO generating agents. Fifty microM SNOC alone had no effect on the cell viability. However, pretreatment with 50 microM SNOC as well as simultaneous application of 50 microM SNOC with NMDA inhibited cell death induced by NMDA. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO, interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.     

Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons

We investigated in rat hippocampus neurons whether 4-(aminobutyl)guanidine (agmatine), formed by decarboxylation of L-arginine by arginine decarboxylase and metabolized to urea and putrescine, can modulate the function of N-methyl-D-aspartate (NMDA) receptor channels. In cultured hippocampal neurons studied by whole-cell patch clamp, extracellular-applied agmatine produced a voltage- and concentration-dependent block of NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid nor kainate currents. Analysis of the voltage dependence of the block suggests that agmatine binds at a site located within the NMDA channel pore with a dissociation constant of 952 microM at 0 mV and an electric distance of 0.62. We also tested effects of several agmatine analogs. Arcaine (1,4-butyldiguanidine) also produced a similar voltage-dependent block of the NMDA current, whereas putrescine (1, 4-butyldiamine) had little effect, suggesting that the guanidine group of agmatine is the active moiety when blocking the NMDA channel. Moreover, spermine (an endogenous polyamine) potentiated the NMDA current even in the presence of blocker agmatine or arcaine, suggesting that the guanidine-containing compounds agmatine and arcaine interact with the NMDA channel at a binding site different from that of spermine. Our results indicate that in hippocampal neurons agmatine selectively modulates the NMDA subclass of glutamate receptor channels mediated by the interaction between the guanidine group and the channel pore. The results support other data that agmatine may function as an endogenous neurotransmitter/neuromodulator in brain.     

Direct effects of metabotropic glutamate receptor compounds on native and recombinant N-methyl-D-aspartate receptors

The actions of glutamate in the central nervous system are mediated through interaction with fast activating ionotropic receptors and G protein-coupled metabotropic glutamate receptors (mGluRs). Studies of these receptors have relied on the availability of agonists and antagonists selective for each receptor class. Compounds that were thought to be selective for mGluRs have been extensively used to study the role of these receptors in the brain. Their use has implicated mGluRs in a wide range of physiological and pathological processes including the modulation of N-methyl-D-aspartate (NMDA) receptors and NMDA receptor-dependent processes. We report that some of the most commonly used mGluR compounds act as antagonists on NMDA receptors at concentrations commonly used to activate or block mGluRs. In addition, several of the drugs also act as agonists at higher concentrations due at least in part to high levels of contaminant amino acids. Our results indicate that caution should be used when using these drugs to study the roles of mGluRs in various NMDA-dependent processes. The antagonist effects were dependent on the concentration of the NMDA receptor coagonists, preventing reappraisal of previously published work.     

Characterization of neuroprotection from excitotoxicity by moderate and profound hypothermia in cultured cortical neurons unmasks a temperature-insensitive component of glutamate neurotoxicity

Although profound hypothermia has been used for decades to protect the human brain from hypoxic or ischemic insults, little is known about the underlying mechanism. We therefore report the first characterization of the effects of moderate (30 degrees C) and profound hypothermia (12 degrees to 20 degrees C) on excitotoxicity in cultured cortical neurons exposed to excitatory amino acids (EAA; glutamate, N-methyl-D-aspartate [NMDA], AMPA, or kainate) at different temperatures (12 degrees to 37 degrees C). Cooling neurons to 30 degrees C and 20 degrees C was neuroprotective, but cooling to 12 degrees C was toxic. The extent of protection depended on the temperature, the EAA receptor agonist employed, and the duration of the EAA challenge. Neurons challenged briefly (5 minutes) with all EAA were protected, as were neurons challenged for 60 minutes with NMDA, AMPA, or kainate. The protective effects of hypothermia (20 degrees and 30 degrees C) persisted after rewarming to 37 degrees C, but rewarming from 12 degrees C was deleterious. Surprisingly, however, prolonged (60 minutes) exposures to glutamate unmasked a temperature-insensitive component of glutamate neurotoxicity that was not seen with the other, synthetic EAA; this component was still mediated via NMDA receptors, not by ionotropic or metabotropic non-NMDA receptors. The temperature-insensitivity of glutamate toxicity was not explained by effects of hypothermia on EAA-evoked [Ca2+]i increases measured using high- and low-affinity Ca2+ indicators, nor by effects on mitochondrial production of reactive oxygen species. This first characterization of excitotoxicity at profoundly hypothermic temperatures reveals a previously unnoticed feature of glutamate neurotoxicity unseen with the other EAA, and also suggests that hypothermia protects the brain at the level of neurons by blocking, rather than slowing, excitotoxicity.     

Chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptor polypeptide subunits in mouse cortical neurons in culture

The goal of this study was to determine whether chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptors (NMDAR) was associated with an augmentation of the NMDAR polypeptide subunits in the mammalian cortical neurons. The results show that chronic ethanol treatment produced an increase in the R1 and R2B polypeptide subunits. The R2A subunit was not expressed in these neurons. Chronic NMDAR antagonist ((+)-3-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)) treatment also increased the R1 and R2B polypeptide subunits. A similar increase was observed when ethanol and CPP were used in combination. Binding studies using [3H]MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclophenptan-5,10-imine maleate), a noncompetitive NMDAR antagonist, confirmed that concomitant exposure of ethanol and CPP up-regulated the NMDAR. Our results demonstrate for the first time that chronic ethanol treatment increased the NMDA receptor polypeptide subunit synthesis and that it was associated with an increase in [3H]MK-801 binding sites.     

On the mechanism of GABA-induced currents in cultured rat cortical neurons

We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by gamma-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl- concentration ([Cl-]i). In current-clamp experiments in the presence of 26 mM HCO3- the application of 50 microM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50-200 microM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO3-. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO3--free saline, indicating a contribution from HCO3-. The reversal potential of the GABA-induced currents (EGABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO3- was -73 mV, while it was shifted to -59 mV in the presence of HCO3-. Combined patch-clamp and microfluorimetric measurements using the Cl--sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl-]i in 54% (n=13) of the neurons. We conclude that this increase of [Cl-]i in combination with the efflux of HCO3- results in a shift of EGABA above the resting membrane potential that gives rise to GABA-mediated depolarizations.     

Vitamin E prevents apoptosis in cortical neurons during hypoxia and oxygen reperfusion

Cerebral ischemia followed by oxygen reperfusion induces apoptosis in hippocampal neurons in stroke-prone spontaneously hypertensive rats (SHRSP) but not in Wistar Kyoto rats (WKY). The overproduction of oxygen-free radicals that occurs in the tissues of SHRSP is implicated in reoxygenation injury after hypoxia. Antioxidants inhibit reoxygenation injury in hippocampal slices, and temporal cortices in Alzheimer's disease increase sensitivity to oxygen-free radicals. Because this sensitivity may contribute to the development of the disease, we have studied hypoxia and oxygen reperfusion using cortical neurons isolated from WKY and SHRSP (at 15 days of gestation). We have tried to determine whether cortical neurons are damaged under these conditions, and whether neurons from SHRSP are more vulnerable than those from WKY. We have tried also to verify whether neuronal damage is minimized by vitamin E using the following techniques: (a) Trypan blue staining, (b) in situ staining of apoptosis, (c) ultrastructural examination, and (d) measurement of lactic dehydrogenase (LDH) activity in the bathing medium. Furthermore, we have examined the mechanisms involved in the development of neuronal damage and have studied ways of minimizing it. We demonstrated that 36 hours of hypoxia significantly increased the rate of cell death in SHRSP (p < 0.01), although 12 to 24 hours of hypoxia did not increase cell death in either WKY or SHRSP. In addition, 6 to 36 hours of hypoxia and 1.5 to 5 hours of oxygen reperfusion heavily damaged cells of both WKY and SHRSP, and most became apoptotic or necrotic. In contrast, cells incubated with 50 to 300 microg/ml of vitamin E remained intact, although 10 to 20 microg/ml of vitamin E did not totally preserve the cells. Moreover, vitamin E protected the neurons from high concentrations of sodium nitroprusside (nitric oxide donor) in a dose-dependent manner. Vitamin E, when added to the cells, increased in concentration in a time-dependent manner over a 24-hour period and in a dose-dependent manner below 200 microg/ml, and it was detected mostly in the mitochondria. We also demonstrated that serial treatments with allopurinol (a xanthine oxidase inhibitor) or superoxide dismutase preserved neurons during hypoxia and oxygen reperfusion. These data indicate that SHRSP neurons are weaker than WKY neurons in long-term hypoxia; oxygen radical generation occurs in the early minutes after reperfusion, and then the oxygen-free radicals cause heavy damage to the cells; and antioxidants including vitamin E react with the radicals, thereby preventing apoptosis and necrosis. Therefore, antioxidants appear to be the most important agents in lowering oxygen-free radical damage in cortical neurons.