Biomedical subjects

P M Beart

Publications and source records attributed to P M Beart.

At least 19 recordsLinked to original sources

PubMed1998

Regional distribution of low affinity kainate receptors in brain of Macaca fascicularis determined by autoradiography using [3H](2S,4R)-4-methylglutamate.

Binding of [3H](2S,4R)-4-methylglutamate, a novel low affinity kainate receptor agonist, was studied in brain sections of a Macaca fascicularis monkey. In cerebellar sections, [3H](2S,4R)-4-methylglutamate bound to a single population of sites (KD = 20 nM) and was inhibited by various glutamate receptor ligands: kainate > 6-cyano-7-nitroquinoxaline-2,3-dione > L-glutamate >> AMPA. (S)-5-lodowillardiine and (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA), drugs selective for the GluR5 subunit, displaced 50% and 40% of binding, respectively. Autoradiography revealed topographic binding of [3H](2S,4R)-4-methylglutamate. Binding in cortex was highest in layer 5 and restricted to CA2/3 in hippocampus. Levels of binding were high in septum and hypothalamus. Moderate densities of binding were found in caudate-putamen, cerebellar granular layer, nucleus tractus solitarius, cuneate nucleus and area postrema. Binding in spinal cord was concentrated in dorsal horn. [3H](2S,4R)-4-Methylglutamate shows differential binding throughout primate brain and is a valuable new ligand for low affinity kainate receptors.

Animals↗

PubMed1998

Mice with a homozygous null mutation for the most abundant glutathione peroxidase, Gpx1, show increased susceptibility to the oxidative stress-inducing agents paraquat and hydrogen peroxide.

Glutathione peroxidases have been thought to function in cellular antioxidant defense. However, some recent studies on Gpx1 knockout (-/-) mice have failed to show a role for Gpx1 under conditions of oxidative stress such as hyperbaric oxygen and the exposure of eye lenses to high levels of H2O2. These findings have, unexpectedly, raised the issue of the role of Gpx1, especially under conditions of oxidative stress. Here we demonstrate a role for Gpx1 in protection against oxidative stress by showing that Gpx1 (-/-) mice are highly sensitive to the oxidant paraquat. Lethality was already detected within 24 h in mice exposed to paraquat at 10 mg.kg-1 (approximately (1)/(7) the LD50 of wild-type controls). The effects of paraquat were dose-related. In the 30 mg.kg-1-treated group, 100% of mice died within 5 h, whereas the controls showed no evidence of toxicity. We further demonstrate that paraquat transcriptionally up-regulates Gpx1 in normal cells, reinforcing a role for Gpx1 in protection against paraquat toxicity. Finally, we show that cortical neurons from Gpx1 (-/-) mice are more susceptible to H2O2; 30% of neurons from Gpx1 (-/-) mice were killed when exposed to 65 microM H2O2, whereas the wild-type controls were unaffected. These data establish a function for Gpx1 in protection against some oxidative stressors and in protection of neurons against H2O2. Further, they emphasize the need to elucidate the role of Gpx1 in protection against different oxidative stressors and in different disease states and suggest that Gpx1 (-/-) mice may be valuable for studying the role of H2O2 in neurodegenerative disorders.

Animals↗

PubMed1998

Type I and II metabotropic glutamate receptors regulate the outflow of [3H]D-aspartate and [14C]gamma-aminobutyric acid in rat solitary nucleus.

Metabotropic glutamate (mGlu) receptors modulating amino acid outflow were examined in a model system in order to further characterize the pharmacological nature of the mGlu receptors involved in viscerosensory processing in the nucleus tractus solitarii. The actions of a number of subtype-selective mGlu receptor agonists and antagonists were monitored on the K+-evoked outflow of [3H]D-aspartate and [14C]gamma-aminobutyric acid (GABA) from superfused slices of rat nucleus tractus solitarii. (+/-)1S,3R-1-Amino-cyclopentane-1,3-dicarboxylate (10-300 microM), produced a concentration-dependent increase in outflow, which was attenuated by a number of phenylglycine antagonists. (2S,3S,4S)-alpha-(Carboxycyclopropyl)-glycine (30-300 microM) had mixed effects on outflow. The type I-selective agonist (RS)-3,5-dihydroxyphenylglycine (300 microM) also increased outflow and these effects were reversed by the type I antagonist (RS)-1-aminoindan-1,5-dicarboxylate (100 microM). Activation of type II mGlu receptors with (2R,4R)-aminopyrrolidine-2,4-dicarboxylate (300 microM), however, decreased outflow, and this effect was antagonized by the type II antagonist LY307452 (200 microM). Interestingly, LY307452 (200 microM) alone, enhanced outflow of [3H]D-aspartate, but not [14C]GABA. Type III mGlu receptors may not be involved in outflow of [3H]D-aspartate and [14C]GABA in the nucleus tractus solitarii, as L-2-amino-4-phosphonobutyrate (30-300 microM) had no effect under the present experimental conditions. These in vitro studies provide new evidence for roles for Type I and II mGlu receptors in viscerosensory processing in nucleus tractus solitarii.

Animals↗

PubMed1998

Transgenic mice expressing human Bcl-2 in their neurons are resistant to 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity.

The protooncogene bcl-2 inhibits neuronal apoptosis during normal brain development as well as that induced by cytotoxic drugs or growth factor deprivation. We have previously demonstrated that neurons of mice deficient in Bcl-2 are more susceptible to neurotoxins and that the dopamine (DA) level in the striatum after systemic 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) administration was significantly lower than in wild-type mice. In the present study we have used transgenic mice overexpressing human Bcl-2 under the control of neuron-specific enolase promoter (NSE-hbcl-2) to test the effects of the neurotoxins 6-hydroxydopamine (6-OHDA) and MPTP on neuronal survival in these mice. Primary cultures of neocortical neurons from normal and transgenic mice were exposed to these dopaminergic neurotoxins. Addition of 6-OHDA resulted in cell death of essentially all neurons from normal mice. In contrast, in cultures generated from heterozygous NSE-hbcl-2 transgenic mice, only 69% of the cells died while those generated from homozygous transgenic mice were highly resistant and exhibited only 34% cell death. A similar effect was observed with neurons treated with MPP+. Moreover, while the striatal dopamine level after MPTP injections was reduced by 32% in the wild type, the concentration remained unchanged in the NSE-hbcl-2 heterozygous mice. In contrast levels of glutathione-related enzymes were unchanged. In conclusion, overexpression of Bcl-2 in the neurons provided protection, in a dose-dependent manner, against neurotoxins known to selectively damage dopaminergic neurons. This study provides ideas for inhibition of neuronal cell death in neurodegenerative diseases and for the development of efficient neuroprotective gene therapy.

Animals↗

PubMed1998

Kainate-induced apoptosis correlates with c-Jun activation in cultured cerebellar granule cells.

We have investigated the involvement of c-Jun in cell death induced by exposure of primary cultures of murine cerebellar granule cells to the glutamate receptor agonist kainate (KA) and evaluated its possible use as a marker for apoptosis. Using cerebellar granule cell neurones from postnatal day 7 mice, we found that 1 hr exposure to KA (1-1000 microM) induced a concentration-dependent neuronal cell death with characteristic apoptotic morphology, including cell shrinkage, neurite blebbing and DNA fragmentation. In addition KA-induced a concentration-dependent expression of c-Jun mRNA and protein as determined by in situ hybridization and immunocytochemistry respectively. DNA fragmentation was detected using terminal transferase-mediated nick-end (TUNEL) labelling and agarose gel electrophoresis. KA-induced cell death was significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), which shifted the concentration-response curve significantly rightward. The number of apoptotic cell bodies, determined by TUNEL, was also reduced by CNQX (50 microM), with only 15-20% of neurones staining positive after exposure to 1mM KA. In addition, the number of positively stained cells for c-Jun protein and mRNA was substantially reduced by CNQX (50 microM) as determined by random and representative cell counts. These results show for the first time that KA induced apoptotic neuronal death in cultured murine cerebellar granule cells involves the induction of c-Jun mRNA and protein, suggesting the involvement of this immediate early gene in excitotoxic receptor-mediated apoptosis and its potential use as a marker for apoptotic cell death.

Animals↗

PubMed1998

Investigations of non-NMDA receptor-induced toxicity in serum-free antioxidant-rich primary cultures of murine cerebellar granule cells.

A culture system was developed whereby murine cerebellar granule cells were grown under serum-free conditions in chemically defined B27-supplemented neurobasal medium plus depolarizing K+ levels, to allow the investigation of the role of agonists at the kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in glutamate-mediated neurotoxicity. Neurones were killed in a concentration-dependent manner by L-glutamate, kainate and its analogues, domoate and 4-(2-methoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid, but not by (S)-AMPA or (S)-5-fluorowillardiine. Kainate (60% maximal cell death at 1mM) was markedly more toxic than NMDA (40% maximal cell death at 1mM) and was shown to be the predominant cause of excitatory amino acid-induced toxicity in these cells as the neuronal death induced by KA was attenuated by the non-NMDA antagonist CNQX, but not the AMPA antagonist LY293558. This study suggests that serum-free cultures of cerebellar granule cells in B27-supplemented neurobasal medium provide a valuable model system for investigations of the role of the kainate receptor in excitatory amino acid-induced neurodegeneration.

Animals↗

PubMed1998

In vivo microdialysis reveals facilitatory metabotropic glutamate receptors regulating excitatory amino acid release in rat nucleus tractus solitarius.

The functional involvement of metabotropic glutamate receptors in nucleus tractus solitarius of the medulla has been examined by evaluating the action of the selective metabotropic glutamate receptor agonist trans-1-amino-cyclopentane-1,3-dicarboxylate on the extracellular release of L-glutamate and L-aspartate by in vivo microdialysis. Studies were performed in urethane anaesthetized rats, with a microdialysis probe being inserted into the medial nucleus tractus solitarius, which was constantly perfused with artificial cerebrospinal fluid (1 microliter/min). Following an equilibration period, samples were collected for amino acid analysis by HPLC. trans-1-Amino-cyclopentane-1,3-dicarboxylate (30 microM) produced 190% and 500% increases in basal release of L-glutamate and L-aspartate, respectively. trans-1-Amino-cyclopentane-1,3-dicarboxylate-evoked release of L-glutamate and L-aspartate was concentration-dependent (10-100 microM), and was calcium and tetrodotoxin-sensitive. Two metabotropic glutamate receptor antagonists, (+)-alpha-methyl-4-carboxyphenylglycine (200 microM) and (S)-4-carboxyphenylglycine (500 microM), when administered via a microdialysis probe, significantly attenuated the trans-1-amino-cyclopentane-1,3-dicarboxylate-evoked release of both L-glutamate and L-aspartate. Basal release was not altered by these two antagonists. Histological studies verified the location of the probe in the nucleus tractus solitarius. These studies provide the first evidence using in vivo microdialysis of functional metabotropic glutamate receptors, which seem to be facilitatory pre- and postsynaptically located receptors, in the rat nucleus tractus solitarius. On the basis of our findings, metabotropic glutamate receptors are likely to play key modulatory roles in regulating transmitter release.

Animals↗

PubMed1998

A reliable procedure for comparison of antioxidants in rat brain homogenates.

Lipid peroxidation is a major consequence of oxidative stress and an important cause of neuronal damage in ischaemic injuries and neurodegenerative disorders such as Parkinson's disease. Recent research has focused on the development of antioxidant drugs which may delay or minimize neurodegeneration. Rapid and reliable assays are therefore necessary in order to evaluate novel antioxidant compounds. A widely adopted method for measurement of lipid peroxidation is the thiobarbituric acid reacting substances (TBARS) assay. Several variations of this method have appeared in the literature, some of which have been tested by us without success. We have therefore established a reliable procedure which takes into account the most important factors previously found to influence the TBARS method. Briefly, various concentrations of drug were added to rat brain homogenates (10% w/v in 20 mM Tris-HCl buffer, pH 7.4) and incubated at 37 degrees C for 10 min before addition of ammonium ferric sulphate (100 or 1000 microM) and a further incubation at 37 degrees C for 30 min. Proteins were then precipitated with 8.1% sodium dodecyl sulphate, the reaction stopped with 20% acetic acid, and the samples were then centrifuged for 15 min. Aliquots of supernatant were added to an equal volume of thiobarbituric acid (0.8%), samples were heated at 95 degrees C for 30 min, and then cooled on ice before reading at 532 nm. The present adaptation represents a simple and highly reproducible assay which does not require difficult extraction procedures with hazardous chemicals and results in a stable chromagen. The method has been evaluated using a number of structurally distinct antioxidants and iron chelators. IC50 values (microM) for percentage inhibition of TBARS formation were as follows: desferroxamine (1.1), U83836E (1.7), butylated hydroxytoluene (13), U74500A (20), LY231617 (22), idebenone (89), and Trolox (110). This order of potency was comparable to that found with a commercially available, but expensive kit designed to specifically measure malondialdehyde (Spearman's rank correlation coefficient, p < 0.01).

Animals↗

PubMed1998

Cyclothiazide and AMPA receptor desensitization: analyses from studies of AMPA-induced release of [3H]-noradrenaline from hippocampal slices.

1. Responses in brain produced by the activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) subtype of ionotropic receptor for L-glutamate are often rapidly desensitizing. AMPA-induced desensitization and its characteristics, and the potentiating effect of cyclothiazide were investigated in vitro by analysing AMPA-induced release of [3H]-noradrenaline from prisms of rat hippocampus. 2. AMPA (1-1000 microM) stimulated the release of [3H]-noradrenaline in a concentration-dependent manner that was both calcium-dependent and tetrodotoxin-sensitive, and attenuated by the AMPA-selective antagonists, NBQX (1 and 10 microM), LY 293558 (1 and 10 microM) and GYKI 52466 (10 and 30 microM). 3. By use of an experimental procedure with consecutive applications of AMPA (100 microM, 28 min apart), the second response was reduced, indicative of receptor desensitization, and was reversed by cyclothiazide in a concentration-dependent manner (1-300 microM). The concentration-response curve for AMPA-induced release of [3H]-noradrenaline was shifted leftwards, but the reversal by cyclothiazide of the desensitized response was partial and failed to reach the maximal response of the first stimulus. 4. Observations made with various schedules of cyclothiazide application indicated that the initial AMPA-evoked response was already partially desensitized (150% potentiation by 100 microM cyclothiazide) and that the desensitization was not likely to be due to a time-dependent diminution and was longlasting (second application of cyclothiazide was ineffective). 5. Co-application of a number of drugs with actions on second messenger systems, in association with the second AMPA stimulus, revealed significant potentiation of the AMPA-induced release of [3H]-noradrenaline: forskolin (10 microM, +78%), Rp-cAMPS (100 microM, +65%), Ro 31-8220 (10 microM, +163%) and thapsigargin (100 pM, + 161%). 6. The AMPA receptor-mediated response regulating the release of [3H]-noradrenaline from rat hippocampal slices was desensitized and cyclothiazide acted to reverse partially the desensitization in a concentration-dependent manner. Since the time-course of desensitization was longer lasting than that noted in previous electrophysiological studies, multiple events may be involved in the down-regulation of AMPA receptor activity including receptor phosphorylation and depletion of intracellular Ca2+ stores.

Animals↗

PubMed1998

Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1.

Recent evidence suggests that neuronal apoptosis is the consequence of an inappropriate reentry into the cell cycle. Expression of the cell cycle gene cyclin D1, a G1-phase cell cycle regulator, was examined in primary cultures of murine cerebellar granule cells (CGCs) during kainate (KA)-mediated apoptosis. Using cultures of CGCs, we found that a 24-h exposure to KA (1-3,000 microM) induced a concentration-dependent cell death with neurons exhibiting characteristic apoptotic morphology and extensive labeling using the terminal transferase-mediated nick end-DNA labeling (TUNEL) method. KA induced a time- and concentration-dependent increase in expression of cyclin D1 as determined by immunocytochemistry and western blot analysis. KA-induced apoptosis and cyclin D1 expression exhibited a similar concentration dependence and were significantly attenuated by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (50 microM), indicating a KA receptor-mediated effect. Here we present evidence for the first time that KA-induced apoptosis in cultured CGCs involves the induction of cyclin D1, suggesting its involvement in excitotoxic receptor-mediated apoptosis.

Animals↗

PubMed1997

Effects of the CCK(A) receptor antagonists SR 27897B and PD140548 on baroreflex function in conscious rats.

Since the cardiovascular effects of cholecystokinin (CCK) seem to particularly involve the A ('peripheral') subtype of CCK (CCK[A]) receptor, we examined the actions of two novel, highly selective CCK(A) receptor antagonists, PD140548 (N-alpha-methyl-N[(tricyclo[3.3.1.1(3,7)]dec-2-yloxy)carbony l]-L-tryptophyl]-D-3-(phenylmethyl)-beta-alanine) and SR 27897B (1-[[2-(4-(2-chlorophenyl)thiazol-2-yl)aminocarbonyl]acetic acid) on CCK-induced alterations in blood pressure and heart rate, and on the baroreceptor reflex in the conscious, instrumented rat. CCK (2 microg, i.v.) produced a pressor response and biphasic effects on heart rate involving an initial bradycardia followed by a pronounced tachycardia. Administration of PD140548 (10 mg/kg, i.v.) and SR 27897B (0.6 mg/kg, i.v.) significantly inhibited the pressor effects of CCK (35 and 47%, respectively), whilst reversing the bradycardic responses to a tachycardia. The CCK(A) receptor antagonists had different effects on the baroreceptor heart rate reflex since only PD140548 caused a significant increase in the gain or sensitivity of the reflex. This effect of PD140548 on gain is likely to occur via a central mechanism and may reflect the increased lipophilicity of PD140548 relative to SR 27897B. Overall, these investigations provide new evidence for the involvement of the CCK(A) receptor in cardiovascular regulation.

Animals↗

PubMed1997

Development and survival of rat embryonic mesencephalic dopaminergic neurones in serum-free, antioxidant-rich primary cultures.

Mesencephalic dopaminergic neurones typically require the presence of serum for their survival in culture. However, the present study, outlines how neurones from the rat ventral mesencephalon (E14-16) were successfully cultured in serum-free, antioxidant-rich Neurobasal medium supplemented with B27 components. Moreover, immunostaining with mouse monoclonal microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) from day 1 to 7 in vitro revealed these cultures were primarily neuronal (80-95%). Additionally, immunostaining with tyrosine hydroxylase (TH) revealed these cultures contained a relatively constant population of TH-positive neurones (5%) which were presumed to be dopaminergic. These primary cultures offer considerable advantages for the study of mesencephalic, TH-positive, dopaminergic neurones under conditions where milieu can be readily manipulated in the virtual absence of glia and without the confounding influence of serum.

Animals↗

PubMed1997

Histochemistry in rat brain and spinal cord with an antibody directed at the cholecystokininA receptor.

Various neurobiological evidence indicates that A-subtype cholecystokinin (CCKA) receptors are widely distributed through the mammalian neuroaxis despite the sparse localization found by receptor autoradiography. To address this paradox, immunohistochemistry has been performed in rat brain and spinal cord using an antibody directed at a portion of the amino terminal sequence of the CCKA receptor. Immunoreactivity, visualised using diaminobenzidine, was widely and topographically distributed being most concentrated in medulla and spinal cord. Many forebrain areas contained specifically labelled neurones, notably the nucleus accumbens, septum, stria terminalis, habenula, substantia nigra, ventral tegmental area and lateral geniculate nucleus. In medulla, heavily labelled perikarya were found in parabrachial and trigeminal nuclei, while in spinal cord immunoreactivity was localized in dorsal horn. Localization of immunoreactivity was consistent with the reported distribution of CCKA receptor-mediated mechanisms. Our observations represent the first attempt to describe the localization of the CCKA receptor in brain using immunohistochemistry and support its wide functional involvement in the central nervous system.

Animals↗

PubMed1997

Cholecystokinin-GABA interactions in rodent cortex: analyses of cholecystokinin effects on K(+)- and L-glutamate-induced release of [3H]GABA from rat cortical slices and cultured mouse cortical neurones.

Neurones of the cerebral cortex immunoreactive for the neuropeptide, cholecystokinin (CCK), also invariably contain GABA. Hence CCK is believed to modulate some aspect of GABAergic synaptic activity. The present study therefore investigated the effects of CCK on basal, K(+)- and L-glutamate-induced release of [3H]GABA from slices of rat neocortex and cultured murine neocortical neurones. Rat neocortical prisms loaded with [3H]GABA (10 nM) were superfused with Krebs-Henseleit buffer and stimulated twice (S1 and S2, 2 min) with K+ (30 mM). Release associated with each stimulus was measured and expressed relative to basal release (R1 and R2). The effects of non-selective and CCKB selective agonists, CCK-8S and CCK-4, respectively, on basal and K(+)-induced release of [3H]GABA were subsequently assessed by alternately including the peptides in S2 and comparing R2/R1 and S2/S1 ratios to control experiments. Contrary to previous findings, CCK-8S (30 nM-1 microM) and CCK-4 (0.3 nM-1 microM) failed to influence basal or K(+)-induced release. In similar experiments, murine cortical neurones superfused with HEPES balanced salt buffer, released exogenous [3H]GABA upon stimulation (1 min) with either K+ (55 mM) or L-glutamate (30 microM). However, CCK-8S, CCK-4 (both 300 nM-1 microM) and the CCKB selective antagonist, L365,260 (1 microM), failed to influence basal, K(+)- or L-glutamate-induced release of [3H]GABA from these neurones when included in S2. These data therefore do not support the postulate that CCK acting via CCKA or CCKB receptors modulates release of GABA under the present experimental conditions. GABA-CCK interactions were not specifically studied because only L-glutamate (30 microM) significantly elevated release of CCK-like immunoreactivity (115% above basal) in murine cortical neurones: basal release of CCK was estimated to be 7 and 11 pM from neurones and slices, respectively. Further studies employing more rigorous stimulation and perhaps examining endogenous GABA release are necessary to fully investigate the co-release of CCK and GABA.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Apoptosis induced via AMPA-selective glutamate receptors in cultured murine cortical neurons.

We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [(S)-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to (S)-AMPA (0.01-1,000 microM) induced concentration-dependent neuronal cell death (EC50 = 3 +/- 0.5 microM) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. (S)-AMPA induced a delayed cell death with DNA fragmentation occurring in approximately 50% of cells at concentrations between 100 and 300 microM detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by (S)-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 microM) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), yielding EC50 values of 73 +/- 5 and 265 +/- 8 microM, respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 microM). The number of apoptotic nuclei induced by 300 microM (S)-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 microg/ml) also inhibited (S)-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

On the distribution of cholecystokinin B receptors in monkey brain.

In view of recent evidence for a role for the B subtype of cholecystokinin (CCKB) receptor in panic and anxiety, the distribution of CCKB receptors in the forebrain of a Rhesus macaca monkey was examined by receptor autoradiography employing [125I]D-Tyr25(Nleu28,31)-CCK25-33S. CCKB receptors were widely and topographically distributed in cortex. Other structures with notable labelling included the basal ganglia, presubiculum, amygdala, mamillary bodies, cerebellar cortex and pineal gland. The distribution of CCKB receptors further supports roles for this peptide in behavioural processes.

Animals↗

PubMed1996

(S)-5-fluorowillardiine-mediated neurotoxicity in cultured murine cortical neurones occurs via AMPA and kainate receptors.

We have examined the neurotoxic effects of kainate, (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and the novel AMPA-receptor preferring agonist (S)-5-fluorowillardiine in murine cultured cortical neurones. Kainate induced > 90% cell death (EC50 65 microM) and (S)-AMPA only about 50% cell death (EC50 3.1 microM), both in a monophasic dose-dependent manner. (S)-5-Fluorowillardiine also killed > 90% of neurones, however, in a biphasic dose-dependent manner (EC50 0.70 and 170 microM). Additionally, the neurotoxic effects of (S)-AMPA and (S)-5-fluorowillardiine (high-affinity component) were attenuated by the AMPA receptor antagonists LY293558 ((3,S,4aR, 6R,8aR)-6[2h91 H-tetrazol-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinol ine- 3-carboxylic acid). A component of kainate and (S)-5-fluorowillardiine (low-affinity component) neurotoxicity was blocked by the low-affinity kainate receptor antagonist NS-102 (5-nitro-6,7,8,9-tetrahydrobenzo[g]indole-2,3-dione-3-oxime). We have shown that both kainate and (S)-AMPA can effect substantial cell death in cortical neurones and that the novel agonist (S)-5-fluorowillardiine exerts its excitotoxicity through both AMPA- and kainate-preferring receptors.

Alanine↗