6-cyano-7-nitroquinoxaline-2-3-dione and Ischemia

Alterations of the enteric glutamatergic transmission may underlay changes in the function of myenteric neurons following intestinal ischemia and reperfusion (I/R) contributing to impairment of gastrointestinal motility occurring in these pathological conditions. The aim of the present study was to evaluate whether glutamate receptors of the NMDA and AMPA/kainate type are involved in myenteric neuron cell damage induced by I/R. Primary cultured rat myenteric ganglia were exposed to sodium azide and glucose deprivation (in vitro chemical ischemia). After 6 days of culture, immunoreactivity for NMDA, AMPA and kainate receptors subunits, GluN(1) and GluA(1-3), GluK(1-3) respectively, was found in myenteric neurons. In myenteric cultured ganglia, in normal metabolic conditions, -AP5, an NMDA antagonist, decreased myenteric neuron number and viability, determined by calcein AM/ethidium homodimer-1 assay, and increased reactive oxygen species (ROS) levels, measured with hydroxyphenyl fluorescein. CNQX, an AMPA/kainate antagonist exerted an opposite action on the same parameters. The total number and viability of myenteric neurons significantly decreased after I/R. In these conditions, the number of neurons staining for GluN1 and GluA(1-3) subunits remained unchanged, while, the number of GluK(1-3)-immunopositive neurons increased. After I/R, -AP5 and CNQX, concentration-dependently increased myenteric neuron number and significantly increased the number of living neurons. Both -AP5 and CNQX (100-500 µM) decreased I/R-induced increase of ROS levels in myenteric ganglia. On the whole, the present data provide evidence that, under normal metabolic conditions, the enteric glutamatergic system exerts a dualistic effect on cultured myenteric ganglia, either by improving or reducing neuron survival via NMDA or AMPA/kainate receptor activation, respectively. However, blockade of both receptor pathways may exert a protective role on myenteric neurons following and I/R damage. The neuroprotective effect may depend, at least in part, on the ability of both receptors to increase intraneuronal ROS production.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cell Count; Cell Survival; Cells, Cultured; Excitatory Amino Acid Antagonists; Ganglia; Glucose; Immunohistochemistry; Ischemia; Male; Myenteric Plexus; Neurons; Rats; Reactive Oxygen Species; Receptors, AMPA; Receptors, Ionotropic Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Reperfusion Injury; Sodium Azide

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Anesthetics, Local; Animals; Bicuculline; Caffeine; Calcium; Cell Count; Cells, Cultured; Cerebral Cortex; Deoxyadenosines; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Fura-2; GABA Antagonists; gamma-Aminobutyric Acid; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Vitro Techniques; Ischemia; Membrane Potentials; Microscopy, Confocal; Microtubule-Associated Proteins; Neurons; Patch-Clamp Techniques; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrodotoxin; Time Factors; Triazines; Tritium; Xanthines

Neuronal activity is accompanied by transmembranous ion fluxes that cause cell volume changes. In whole mounts of the guinea pig retina, application of glutamate resulted in fast swelling of neuronal cell bodies in the ganglion cell layer (GCL) and the inner nuclear layer (INL) (by approximately 40%) and a concomitant decrease of the thickness of glial cell processes in the inner plexiform layer (IPL) (by approximately 40%) that was accompanied by an elongation of the glial cells, by a thickening of the whole retinal tissue, and by a shrinkage of the extracellular space (by approximately 18%). The half-maximal effect of glutamate was observed at approximately 250 mum, after approximately 4 min. The swelling was caused predominantly by AMPA-kainate receptor-mediated influx of Na+ into retinal neurons. Similar but transient morphological alterations were induced by high K+ and dopamine, which caused release of endogenous glutamate and subsequent activation of AMPA-kainate receptors. Apparently, retinal glutamatergic transmission is accompanied by neuronal cell swelling that causes compensatory morphological alterations of glial cells. The effect of dopamine was elicitable only during light adaptation but not in the dark, and glutamate and high K+ induced strong ereffects in the dark than in the light. This suggests that not only the endogenous release of dopamine but also the responsiveness of glutamatergic neurons to dopamine is regulated by light-dark adaptation. Similar morphological alterations (neuronal swelling and decreased glial process thickness) were observed in whole mounts isolated immediately after experimental retinal ischemia, suggesting an involvement of AMPA-kainate receptor activation in putative neurotoxic cell swelling in the postischemic retina.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids; Animals; Aspartic Acid; Cell Size; Circadian Rhythm; Cytochalasin D; Cytoskeleton; Dicarboxylic Acids; Dopamine; Eye Proteins; Glutamic Acid; Guinea Pigs; Ischemia; Kainic Acid; N-Methylaspartate; Neuroglia; Neurons; Nocodazole; Potassium; Pyrrolidines; Receptors, AMPA; Receptors, Kainic Acid; Retina; Retinal Ganglion Cells; Synaptic Transmission; Xanthenes

The present experiments were carried out on three types of neurone in primary rabbit retinal cultures. One cell-type, bipolar neurones, have glutamate APB-type metabotropic receptors and can be identified by the presence of thetaPKC-immunoreactivity. The other two cell-types are primarily amacrine cells and can be 'stained' for the localisation of GABA immunoreactivity or for serotonin taken up from the medium. Most of the serotonin-accumulating and GABA-containing neurones contain glutamate kainate-type receptors. Exposure of the cultures to treatment of kainate (50 microM) or experimental ischaemia (8 h followed by 16 h reoxygenation) produced essentially similar findings. The serotonin-accumulating and GABA cells were affected as they were drastically reduced in numbers while the numbers of thetaPKC-containing cells were unaffected. Inclusion of the kainate/AMPA antagonist CNQX (100 microM) or melatonin (100 microM) to the medium during kainate or ischaemia treatments largely prevented the detrimental influences on the serotonin-accumulating and GABA cells. It is concluded that during experimental ischaemia excessive glutamate is released to influence cells which contain kainate and APB-type receptors. However, only the neurones containing the kainate receptors are negatively affected with the generation of free radicals. Melatonin or CNQX protects against this effect by scavenging free radicals or acting at the receptor level, respectively.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Excitatory Amino Acid Antagonists; Ischemia; Kainic Acid; Melatonin; Neurons; Rabbits; Receptors, Kainic Acid; Retina; Retinal Vessels

The aim of the described experiments was to use immunohistochemistry to visualize the release of GABA from specific retinal amacrine cells following ischaemia and to establish the involvement of defined glutamatergic receptors. In initial experiments, rabbit retinas were exposed in vitro to excitatory amino acid agonists alone or in combination with a putative antagonist, or in physiological solution lacking oxygen and glucose, or in solution containing potassium cyanide for 45 min at 37 degrees C. The nature of the GABA immunoreactivity was then examined by immunohistochemistry. In other in vitro experiments, retinas were first allowed to accumulate exogenous serotonin before exposing the tissues to the combinations as described. These tissues were then processed immunohistochemically for the localization of serotonin. In yet other experiments, the intraocular pressure of a rabbit's eye was raised to about 110 mmHg for 60 min and a reperfusion time of 45 min allowed before dissecting the retina and processing for the localization of GABA immunoreactivity. The other eye served as a control. Of the excitatory amino acid agonists tested, only N-methyl-D-aspartate, kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid caused a change in the GABA immunoreactivity. The N-methyl-D-aspartate effect was specifically antagonized by dizocilpine maleate, dextromethorphan and memantine, and was characterized by a reduction in the number of GABA-immunoreactive perikarya. The GABA "staining" in the inner plexiform layer also appeared as four clear bands. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-induced effects were both antagonized by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione and partially by kynurenic acid at the concentrations used. Here, the amount of GABA-positive perikarya was greatly reduced and three immunoreactive bands appeared in the inner plexiform layer. However, for low concentrations of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid four GABA-immunoreactive bands could be identified in the inner plexiform layer. The normal GABA immunoreactivity of the inner plexiform layer also appeared to be in defined bands in retinas which received an ischaemic insult either by reducing the availability of glucose and oxygen, exposing the tissue to potassium cyanide or raising the intraocular pressure of an eye. In these cases the number of GABA-positive perikarya was also reduced. Only alpha-amino-3-hy

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Aminobutyrates; Animals; Cycloleucine; Dextromethorphan; Eye Proteins; gamma-Aminobutyric Acid; Glucose; Glutamates; Glutamic Acid; Intraocular Pressure; Ischemia; Kainic Acid; Kynurenic Acid; Memantine; N-Methylaspartate; Oxygen; Potassium Cyanide; Quinoxalines; Rabbits; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Retina; Retinal Vessels; Serotonin

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Chickens; Dizocilpine Maleate; Edema; Electron Transport; Glycolysis; Hypoglycemia; Hypoxia; Iodoacetates; Iodoacetic Acid; Ischemia; N-Methylaspartate; Potassium Cyanide; Quinoxalines; Receptors, N-Methyl-D-Aspartate; Retina; Tetrodotoxin