The mechanisms underlying neuronal injury caused by hypoxia and ischemia are not understood. Calcium ion influx through the N-methyl-D-aspartate (NMDA) receptor is thought to be important in the pathogenesis of this injury as this is key to a delayed form of neuronal injury observed in vitro. Passive redistribution of chloride ions following sodium entry through NMDA receptors is thought to be involved in the pathogenesis of an immediate form of neuronal injury in vitro. This proposal to study the calcium dependent regulation of NMDA receptors has five main objectives: The first is to further characterize an extracellular calcium ion dependent desensitization of the NMDA receptor, since this may be a protective mechanism from exposure to excessive amounts of excitatory amino acids. These experiments will involve the use of the photolabile compound Nitr-5 in order to determine the site of action of calcium (intracellular or extracellular). Rapid drug delivery to outside-out membrane patches from postnatal rat hippocampal neurons grown in culture will be used to determine whether or not NMDA desensitization on patches is also calcium dependent. It is expected that these experiments will lead to a further delineation of the site of action of calcium and of the channel correlate of this process. A second objective is to further characterize a voltage dependent chloride current in hippocampal neurons since this current may be important in immediate neuronal injury. Preliminary studies reveal a block of this current by activators of protein kinase C, making this current an ideal candidate for a positive control in further studies of the modulation of NMDA responses by protein kinase C. Because of the suspicion that an observed increase in NMDA responsiveness may be caused by a calcium dependent messenger system, the third objective is to study the intracellular calcium ion triggered messenger regulation of NMDA currents. Using the protein kinase C regulation of the observed chloride current as a positive control, experiments will be designed to study the modulation of NMDA currents by this enzyme system. The last two objectives are to study the influence of modulators of NMDA currents and of the chloride current upon synaptic transmission and upon NMDA toxicity.
