The reversibility of early damage caused by excitatory neurotransmitters will be studied in the hippocampal slice preparation. In tissue culture, neuronal death can be prevented if N-methyl-D-asparate (NMDA) receptor antagonists are applied during a critical period after exposure to excitotoxic concentration of glutamate. The injury that eventually results in neuronal degeneration appears to be a prolonged process that depends on sustained activation of NMDA receptors. The hippocampal slice preparation provides unique access to vulnerable adult neurons in vitro. After brief incubation of slices with excitatory amino acids, three markers of early injury will be used to map toxicity while the process is still potentially reversible: 1) regional calcium uptake, 2) regional protein synthesis, and 3) regional Na-K ATPase activity. In preliminary experiments, brief exposure to glutamate agonists produced dose-dependent increases in calcium uptake and sustained decreases in protein synthesis. The first goal of the proposed experiments will be to fully characterize the receptor specificity, time course, and distribution of these early changes associated with excitotoxicity. After exposure to glutamate agonists, NMDA receptor antagonists will be added to the medium to reverse of prevent sustained changes, as has been described in tissue culture. The source of the endogenous transmitter causing sustained NMDA receptor activation after excitotoxic exposure will be determined. Release and reuptake of glutamate from slices will be measured directly. Either increased release or decreased reuptake of transmitter could result in excessive postsynaptic receptor activation. Blockade of glutamate release at the presynaptic terminal by lesions, metabolic inhibitors, and baclofen will be examined for protective effects. Impulse flow in excitatory pathways will be blocked with acute lesions and TTX, to test the hypothesis that sustained NMDA receptor activation is due to excessive activity in excitatory pathways. Experiments are planned to determine which of the postsynaptic effects of NMDA receptor activation produces signs of injury. After brief ischemia in gerbils, administration of NMDA receptor antagonists prevents neuronal death in the hippocampus. This suggests that the sustained activation of NMDA receptors under investigation in the proposed experiments also produces hypoxic- schemic neuronal damage in vivo. Excessive activation of excitatory amino acid receptors may also play a role in the pathophysiology of hypoglycemia and prolonged seizures. An understanding of the reversible events of neuronal injury caused by excitatory transmitters may lead to clinically useful pharmacological therapies in human disease.
