Direct observation of the agonist-specific regional vulnerability to glutamate, NMDA, and kainate neurotoxicity in organotypic hippocampal cultures.
Excessive activation of excitatory amino acid receptors has been implicated in the neuronal degeneration caused by ischemia, hypoglycemia, and prolonged seizures. We have observed directly the time course and regional vulnerability of hippocampal neurons to glutamate receptor-mediated injury in organotypic hippocampal cultures, a preparation which combines accessibility and long-term survival with preservation of regional differentiation and neuroanatomic organization. Cultures were incubated with the fluorescent dye propidium iodide which selectively enters and stains cells only after membrane damage. After 5 to 10 min of a 30-min exposure to kainate (100 microM), large neurons in the hilus of the dentate were first to become brightly fluorescent. Propidium staining subsequently appeared in the other regions of the hippocampus and increased to a maximum over the first 6 h of recovery. NMDA (10 microM) caused propidium staining that was limited to CA1 and the dentate gyrus of the cultures, sparing CA3, consistent with the regions of highest NMDA receptor density in vivo. Glutamate (1 mM) caused a delayed, progressive pattern of staining that began in CA1 (2 to 4 h after exposure), then extended to include CA3 and finally the dentate gyrus over the next 24 h. Release of LDH activity into the media was slower and less sensitive than propidium staining. Histologic degeneration was limited to neurons 24 h after agonist exposure and was consistent with the propidium staining. NMDA, kainate, and glutamate each produced a unique pattern of neuronal injury. Most notably, glutamate had low potency as a toxin and its pattern of neuronal injury was not reproduced by NMDA.
Vornov
J J
JJ
Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
Tasker
R C
RC
Coyle
J T
JT
D000818:Animals; D046508:Culture Techniques; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D005971:Glutamates / Q000494:pharmacology*; D018698:Glutamic Acid; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007608:Kainic Acid / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000473:pathology; D009994:Osmolar Concentration; D011419:Propidium; D013194:Staining and Labeling
D005971:Glutamates; D011419:Propidium; D018698:Glutamic Acid; D016202:N-Methylaspartate; D007770:L-Lactate Dehydrogenase; D007608:Kainic Acid