Biomedical subjects

P M Beart

Publications and source records attributed to P M Beart.

At least 19 recordsLinked to original sources

PubMed2000

Direct visualization of cholecystokinin subtype2 receptors in rat central nervous system using anti-peptide antibodies.

The cholecystokinin receptor, subtype 2 (CCK(2)R), is considered, based on receptor autoradiography, to be the predominant receptor for this peptide transmitter in the mammalian central nervous system. To directly visualize the CCK(2)R we utilized a convenient and sensitive immunohistochemical procedure using antipeptide receptor antibodies raised in rabbits against unique portions of the carboxyl tail and third intracellular loop of the CCK(2)R. Antibodies were characterized by ELISA and Western blotting, and used for immunohistochemistry in rat brain sections. Studies with both antibodies revealed a widespread topographic distribution of CCK(2)R-like immunoreactivity (CCK(2)R-LI) in regions such as cortex, olfactory bulb, nucleus accumbens, septum, striatum, hippocampus, basolateral amygdala, habenula, hypothalamus, thalamus, ventral mesencephalon, inferior colliculus, parabrachial nucleus, pontine nucleus, supercolliculus, red nucleus, subcommisural and occulomotor nucleus, area postrema, solitary, olivary, cochlear, cuneate and trigeminal nuclei and spinal cord dorsal horn in agreement with the results of previous receptor autoradiography.

Amino Acid Sequence↗

PubMed2000

A novel, rapid, computerized method for quantitation of neuronal damage in a rat model of stroke.

Determination of extent of infarction in animal models of cerebral ischemia is most commonly achieved by either classical histology (thionin staining) and light microscopy or staining with 2,3, 5-triphenyltetrazolium chloride (TTC). These techniques have limitations and we now describe a novel technique and its validation for assessment of the neuroprotective activity of AM-36, a novel arylalkypiperazine compound with combined antioxidant and sodium channel blocking activity. AM-36 (1.8 mg/kg i.p.) or vehicle, was administered 30 min, 24 and 48 h after endothelin-1-induced middle cerebral artery occlusion in conscious rats. Rats were killed at 72 h, brains removed and frozen in liquid nitrogen prior to coronal sectioning. Using a simple apparatus relying on basic principles of light propagation and a computerised image analysis system, ischemic damage in unstained slide-mounted sections was clearly visualised and measured. AM-36 significantly reduced the area of infarct in both cortex and striatum. The method was verified by thionin staining, and light microscopy. Linear regression analysis showed a highly significant correlation between methods at 72 h for infarct area in the cortex and striatum. Highly significant correlations between methods were found at 3 and 24 h after ischemia. Our method quickly and clearly delineates areas of damage in a manner superior to conventional staining methods.

Animals↗

PubMed2000

Characterisation of GABA(A) receptors in fetal, neonatal and adult ovine brain: region and age related changes and the effects of allopregnanolone.

Progesterone metabolites acting via GABA(A) receptors suppress central nervous system (CNS) activity. The aim of the present study was to examine binding characteristics of GABA(A) receptors in fetal, newborn and adult sheep brains using [(35)S]TBPS, and to determine the effects of allopregnanolone on this binding. Receptor affinity (K(D)) and density (B(MAX)) in the brainstem were not different in fetal, newborn (1-2 days old) and adult brains. In the hypothalamus K(D) and B(MAX) increased significantly in the fetus between 85 and 128 days gestation, and were then similar to postnatal and adult values. In the frontal cortex K(D) and B(MAX) increased progressively between 85 days and term ( approximately 147 days gestation), and were then not different from postnatal and adult values. The K(i) values for the GABA(A) receptor antagonist picrotoxin was similar at all ages. Allopregnanolone inhibited [(35)S]TBPS binding in the presence of 5 microM GABA, but enhanced binding in the absence of GABA. These results show that (i), functional GABA(A) receptors are present in the fetal brain from at least 85 days gestation; (ii), 3alpha-pregnane steroids modify receptor affinity in the late gestation fetal brain; and (iii) there are region-specific changes in GABA(A) receptor binding parameters. Steroid modulation of the GABA(A) receptor in the fetal brain is likely to influence fetal CNS activity in late gestation.

Aging↗

PubMed2000

Excitotoxic profile of LY339434, a GluR5 agonist, in cultured murine cortical neurons.

The neurotoxic profile of (2S,4R, 6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434), a low-affinity kainate receptor subtype 5 (GluR5) agonist at recombinant human glutamate receptors, was evaluated to investigate the involvement of GluR5 in excitotoxic neuronal death. Murine cortical neurons were exposed to treatments for 24 h and assessed by a cell viability assay and phase-contrast microscopy. LY339434 (1-1000 microM) caused a concentration-dependent decrease in cell viability (EC(50)=11.4+/-1.2 microM) that was only attenuated by (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine (MK-801, 10 microM), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466, 20 microM). Labeling with nucleic acid binding dyes revealed that LY339434 induced few apoptotic-like characteristics. These findings indicate that in cultured murine cortical neurons, LY339434 acts predominantly through N-methyl-D-aspartate (NMDA) receptors rather than GluR5 to effect neuronal death that is rapid and involves predominantly necrosis rather than morphological apoptosis.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2000

Low-affinity kainate receptor agonists induce insult-dependent apoptosis and necrosis in cultured murine cortical neurons.

Overstimulation of ionotropic glutamate receptors leads to excitotoxic neuronal death, which has been implicated in the neurodegeneration of neurological diseases. The present study examined the role of putative low-affinity kainate receptor subtype (GluR5-7) agonists in excitotoxicity in cultured murine cortical neurons. The concentration-dependent decrease in cell viability induced by the agonists kainate (1-1,000 microM) and (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA; 1-1,000 microM) was only attenuated by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466; 20 microM). (S)-5-iodowillardiine (1-1,000 microM)-induced toxicity was attenuated by CNQX (20 microM), GYKI 52466 (20 microM) and MK-801 (10 microM); however, (2S, 4R)-4-methylglutamate (1-120 microM)-induced toxicity was not attenuated by the antagonists. None of the agonists possessed selective actions at GluR5-7. Morphological observations (phase-contrast and fluorescence microscopy) revealed that the agonists induced two distinct patterns of neuronal injury. After 24 hr of treatment, low concentrations of agonists (1-30 microM) produced cellular shrinkage and nuclear granulation consistent with slow, apoptotic-like neuronal death. Pyknotic labeling with the DNA binding dye Sytox green confirmed these apoptotic characteristics, which significantly decreased with increasing concentrations. After 4 hr, increasing concentrations of agonists (100-1,000 microM) induced cellular swelling, with subsequent extracellular debris; labeling with propidium iodide revealed isolated nuclei consistent with the increased involvement of rapid necrosis. Thus, all putative GluR5-7 agonists produced excitotoxicity across a necrotic-apoptotic continuum in murine cortical neuron cultures.

Animals↗

PubMed2000

CCK/dopamine interactions in Fawn-Hooded and Wistar-Kyoto rat brain.

The aim of this study was to compare the actions of CCK neuropeptides within the nucleus accumbens (N.Acc) of alcohol preferring (Fawn-Hooded, FH) and alcohol nonpreferring (Wistar-Kyoto, WKY) rats. CCK-8S (30-300 nM) facilitated the K(+) stimulated release of [(3)H]dopamine (DA) from N.Acc prisms in both rat strains, whereas CCK-4 (30 nM-1 microM) caused a significant decrease of evoked [(3)H]DA in the FH rat only. A scattered distribution of CCK-A and -B receptor immunopositive varicose fibers were visualized throughout the N.Acc of both rat strains along with a topographic distribution of CCK receptor positive cells throughout the ventral mesencephalon.

Alcohol Drinking↗

PubMed2000

Human Bcl-2 protects against AMPA receptor-mediated apoptosis.

Dysfunctions of the (S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of ionotropic receptor for the brain's major excitatory neurotransmitter, L-glutamate, occur in various neurological conditions. We have previously demonstrated that AMPA receptor-mediated excitotoxicity occurs by apoptosis and here examined the influence of the expression of cell death repressor gene Bcl-2 on this excitotoxic insult. Using neuronal cortical cultures prepared from transgenic mice expressing the human Bcl-2 gene, the influence of Bcl-2 on AMPA receptor-mediated neuronal death was compared with that seen with staurosporine and H2O2. At day 6 cultures were exposed to AMPA (0.1-100 microM), and cellular injury was analyzed 48 h after insult using phase-contrast microscopy, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay, and DNA staining with 4,6-diamidino-2-phenylindole and Sytox Green. AMPA produced a concentration-dependent increase in cell death that was significantly attenuated by human Bcl-2. AMPA (3 microM) increased the number of apoptotic nuclei to 60% of control in wild-type cultures, and human Bcl-2 significantly decreased the number of apoptotic nuclei to 30% of AMPA-treated cultures. Human Bcl-2 only provided significant neuroprotection against neuronal injury induced by low concentrations of staurosporine (1-10 nM) and H2O2 (0.1-30 microM) and where neuronal death was by apoptosis, but not against H2O2-induced necrosis. Our findings indicate that overexpression of Bcl-2 in primary cultured neurons protects in an insult-dependent manner against AMPA receptor-mediated apoptosis, whereas protection was not seen against more traumatic insults. This study provides new insights into the molecular therapeutics of neurodegenerative conditions.

Animals↗

PubMed1999

Type I and II metabotropic glutamate receptors mediate depressor and bradycardic actions in the nucleus of the solitary tract of anaesthetized rats.

The potential role of metabotropic glutamate (mGlu) receptors in cardiovascular function in the nucleus of the solitary tract was examined following the microinjection of a number of selective mGlu receptor compounds into this site of anaesthetized rats. The prototypic mGlu receptor selective agonist 1S,3R-1-amino-cyclopentane dicarboxylate elicited depressor and bradycardic actions following microinjection into the nucleus tractus solitarius, which were similar to those produced by L-glutamate. Similarly, decreases in blood pressure and heart rate were observed upon administration of the type I and II selective mGlu receptor agonists, (R,S)-3,5-dihydroxyphenylglycine (DHPG) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC), respectively. These actions of DHPG were selectively attenuated by (+/-)-1-aminoindane-1,5-dicarboxylate, a type I mGlu receptor antagonist, whilst cardiovascular responses to APDC were unaffected by this compound. Interestingly, the proposed type II antagonist, (2S,4S)-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-doic acid, reduced the cardiovascular responses to intra-nucleus tractus solitarius administration of both APDC and DHPG. The type III mGlu receptor agonist, L-2-amino-4-phosphonobutyrate, however, failed to elicit any cardiovascular actions when microinjected into the nucleus tractus solitarius. These studies provide new evidence for functional type I and II mGlu receptors in modulating cardiovascular responses in the nucleus tractus solitarius.

Anesthesia↗

PubMed1999

Excitotoxic injury profiles of low-affinity kainate receptor agonists in cortical neuronal cultures.

Neurotoxic profiles of putative agonists for low-affinity kainate subtypes of L-glutamate receptors (GluR5-7) were determined in cultured cortical neurones. Rank order of neurotoxic potency (microM): (S)-5-iodowillardiine (9) approximately = (2S,4R,6E)-2-amino-4-carboxy-7-(2-naphthyl)hept-6-enoic acid (LY339434, 11) > (2S,4R)-4-methylglutamate (33) > kainate (100) > (RS)-2-amino-3-(hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA, 360). Using ionotropic glutamate receptor antagonists, neurotoxicity induced by kainate, ATPA and (S)-5-iodowillardiine appeared to involve a GluR5-7 component, unlike LY339434 and (2S,4R)-4-methylglutamate. These putative GluR5-7 agonists exhibited complex excitotoxic profiles highlighting the importance of studying native glutamate receptors.

Animals↗

PubMed1999

Cyclothiazide and GYKI 52466 modulate AMPA receptor-mediated apoptosis in cortical neuronal cultures.

In neocortical neuronal cultures, (S)-AMPA caused neurotoxicity which was concentration-dependent, receptor-mediated, slow and apoptotic in nature. (S)-AMPA (3-600 microM) failed to produce rapid neuronal swelling, but morphological observations and monitoring of viability at 24-72 h revealed 50% cell death consistent with apoptosis. (S)-AMPA induced cell shrinkage, neurite blebbing and nuclear condensation. Cyclothiazide (50 and 100 microM), which blocks AMPA receptor desensitization potentiated excitotoxicity with 75% of neurones undergoing slow death. The AMPA-selective antagonist GYKI 52466 (10-50 microM), attenuated (S)-AMPA-mediated neurotoxicity. DNA condensation, a hallmark of apoptosis, was found by labelling neurones with the DNA binding dye 4,6-diamidino-2-phenylindole HCl (DAPI). Gel electrophoresis revealed DNA fragmentation, which was increased by cyclothiazide and reduced by GYKI 52466 and cycloheximide. Overstimulation of the AMPA receptor produces a novel form of neuronal death, which is apoptotic, very slow in nature, and which could contribute to various neuropathologies.

Animals↗

PubMed1999

Type I and II metabotropic glutamate receptor agonists and antagonists evoke cardiovascular effects after intrathecal administration in conscious rats.

1. In the present study, the role of metabotropic glutamate receptors (mGluRs) in central cardiovascular regulation in conscious rats was examined. To this end, agonists and antagonists for type I and II mGluRs were administered intrathecally, and the temporal changes in blood pressure and heart rate were recorded. 2. L-glutamate (1 micromol) and the prototypical mGluR agonist (1S,3R)-ACPD (0.1 and 0. 3 micromol) both increased mean arterial pressure (MAP) and heart rate (HR), implicating functional mGluRs in the spinal cord. The type I mGluR agonist DHPG (0.01 - 0.1 micromol) evoked increases in MAP (max=25+/-5 mmHg) and HR (max=88+/-23 beats min-1). The duration of action, but not the maximum effects, were dose-related and ranged from approximately 10 min to <90 min and 1 min to >90 min for MAP and HR, respectively. 3. The type I/II mGluR agonist CCG-1 (0.1 and 0. 3 micromol) caused smaller, variable increases in MAP and HR of intermediate duration (5 - 20 min), whereas the type II MGluR agonist APDC (0.1 and 1.0 micromol) caused marked, but transient (3 - 5 min), pressor and tachycardic responses. The highest doses of DHPG and CCG-1, but not APDC, also evoked behavioural responses similar to a spontaneous nociceptive behavioural effect reported previously. 4. The type I and II mGluR antagonists (AIDA and LY307452, respectively) were also given approximately 5 min before the administration of the respective type I and II mGluR agonists (DHPG and APDC). Both compounds caused pressor and tachycardic responses, with the effect of AIDA, but not LY307452, returning to control levels before mGluR agonist administration. AIDA significantly attenuated the overall cardiovascular effects of DHPG, while LY307452 significantly attenuated the overall cardiovascular effects of APDC. 5. These results indicate that functional type I and II mGluRs exist in the spinal cord, and that their activation evokes prolonged cardiovascular effects.

Animals↗

PubMed1999

Delayed treatment with AM-36, a novel neuroprotective agent, reduces neuronal damage after endothelin-1-induced middle cerebral artery occlusion in conscious rats.

BACKGROUND AND PURPOSE: AM-36 is a novel arylalkylpiperazine with combined antioxidant and Na(+) channel blocking actions. Individually, these properties have been shown to confer neuroprotection in a variety of in vitro and in vivo animal models of stroke. Preliminary studies have shown that AM-36 is neuroprotective in vivo. The purpose of the present study was to assess the neuroprotective and behavioral outcome after delayed administration of AM-36 in an endothelin-1-induced, middle cerebral artery model of cerebral ischemia in conscious rats. METHODS: Conscious male hooded Wistar rats were subjected to middle cerebral artery occlusion by perivascular microinjection of endothelin-1 via a previously implanted cannula. AM-36 (6 mg/kg IP) or vehicle was administered intraperitoneally 30, 60, or 180 minutes after middle cerebral artery occlusion. Functional outcome was determined 24, 48, and 72 hours after stroke by neurological deficit score, motor performance, and sensory hemineglect tests. Rats were killed at 72 hours, and infarct area and volume were determined by histology and computerized image analysis. RESULTS: Endothelin-1-induced middle cerebral artery occlusion resulted in marked functional deficits and neuronal damage. AM-36 significantly reduced cortical damage when administration was delayed until 30, 60, or 180 minutes after stroke. Interestingly, neuronal damage was time-dependently reduced, with the greatest protection found when AM-36 was administered 180 minutes after stroke. Striatal damage was significantly reduced after treatment with AM-36 at 180 minutes after stroke. Functional outcome paralleled histopathology. Rota-rod performance, sensory hemineglect, and neurological deficit scores returned to preischemia levels in AM-36-treated rats by 72 hours after stroke when administration was delayed by 180 minutes after stroke. CONCLUSIONS: AM-36 potently protects against both neuronal damage and functional deficits even when administered up to 180 minutes after induction of stroke. In fact, the greatest protection was found when administration was delayed by 180 minutes after stroke. The possible mechanisms of action of AM-36 are discussed. The present findings suggest that AM-36 may have great promise in the acute treatment of human stroke.

Animals↗

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↗