Subject(s)
Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Neuroprotective Agents/pharmacology , Ouabain/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, N-Methyl-D-Aspartate/drug effects , Animals , Apoptosis/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , N-Methylaspartate/pharmacology , Neurons/drug effects , Neurons/metabolism , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitorsABSTRACT
The ratio of necrosis to apoptosis and the mechanisms of apoptosis were studied during neurodegeneration induced by glutamate and selective agonists of glutamate receptors - N-methyl-D-aspartate (NMDA) and kainate. Experiments were performed on primary cultures (seven days in vitro) of rat cerebral cortex neurons. Apoptosis and necrosis were identified using a vital fluorescence rapid test with staining with acridine orange and ethidium bromide. Immunocytochemistry in combination with confocal microscopy was used to visualize apoptotic proteins. Agonists (240 min) caused neuron death via both processes, though the proportion of necrotic cells when neurodegeneration was induced by NMDA and kainate was significantly less than when neurodegeneration was induced with glutamate. The neurotoxic effect of 3 mM glutamate was mediated via alpha-amino-3-(3-hydroxy-5-methylisoxazole-4-yl)propionate (AMPA) and kainate receptors, as it was blocked by 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX). Activation of NMDA receptors led to the development of apoptosis without involvement of caspases, due to the direct action of apoptosis-inducing factor (AIF) on neuron nuclei. Activation of AMPA-kainate receptors was accompanied by the development of apoptosis via the caspase-dependent pathway. Thus, these data identified the receptor dependence of the mechanisms of apoptosis during the neurotoxic action of glutamate.
Subject(s)
Apoptosis/drug effects , Cell Nucleus/metabolism , Cerebral Cortex/metabolism , Embryo, Mammalian/metabolism , Glutamic Acid/toxicity , Neurons/metabolism , Receptors, Glutamate/metabolism , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Active Transport, Cell Nucleus/drug effects , Animals , Apoptosis Inducing Factor/metabolism , Caspase 3/metabolism , Cell Nucleus/pathology , Cells, Cultured , Cerebral Cortex/pathology , Dose-Response Relationship, Drug , Embryo, Mammalian/pathology , Excitatory Amino Acid Agonists/toxicity , Excitatory Amino Acid Antagonists/pharmacology , Kainic Acid/pharmacology , N-Methylaspartate/pharmacology , Necrosis/metabolism , Necrosis/pathology , Neurons/pathology , Rats , Rats, Wistar , Tumor Suppressor Protein p53/metabolismABSTRACT
A contribution of necrosis and apoptotis as well as the particular apoptosis pathways in neuro-degeneration induced by glutamate and selective glutamate receptor agonists, NMDA and kainate, were studied. In experiments on primary neuron cultures of 7 days in vitro from embryonic rat cortex, the necrosis and apoptosis were recognized using vital fluorescence acridine orange and ethidium bromide staining. Immunostaining was used to visualize apoptotic peptides such as P53, Cas-3 and AIF. Death of neurons occurred by both necrosis and apoptosis following 240 min 3 mM glutamate, 30 microM NMDA and 30 microM kainate exposure. Quantities of necrotic neurons in the presence of NMDA and kainate were substantially reduced when compared to the glutamate action. The glutamate effects were realized through predominant activation of AMPA- and kainate receptors, since it could be greatly suppressed by 30 microM CNQX. AIF but not Cas-3, was found in a large amount of neurons when apoptosis was evoked by the selective NMDA receptor activation. On the contrary, during apoptosis induced by glutamate and kainate, many cells contained Cas-3 in nuclei rather than the AIF. The data suggest that apoptosis induced by the NMDA receptor activation develops through the caspase-3-independent pathway that involves direct AIF accumulation in nuclei. The AMPA/kainate receptor mediated apoptosis includes the caspase-3-dependent mechanism.