The goal of the proposed research is to understand the effects of volatile anesthetics on the N-methyl-D-aspartate (NMDA) receptor-channel complex and to appreciate the role of these actions in the development and maintenance of the anesthetic state. We have shown that 2 volatile anesthetics (enflurane and halothane) selectively inhibit glutamate-stimulated [3H]MK-801 binding to NMDA receptor ion channels and block NMDA receptor-mediated 45Ca flux into rat brain microvesicles. Moreover, this inhibition is attenuated by the allosteric NMDA modulator, glycine. These findings raise the intriguing possibility that volatile anesthetics depress synaptic transmission at NMDA receptors by disrupting glutamate activation (opening) of the intrinsic ion channel. The hypotheses to be tested are that volatile anesthetics depress excitatory neurotransmission at NMDA receptors by disrupting 1) agonist binding to the glutamate recognition site, 2) ligand binding to the glycine modulatory site, and/or 3) glutamate, glycine, cation and polyamine activation of NMDA receptor ion channels. These hypotheses will be tested using radiolabelled probes for agonist, regulatory and ion channel binding sites on the NMDA receptor complex, as well as direct measurement of receptor-generated intra-cellular calcium signals. Accordingly, the specific aims are to determine the effects of several volatile anesthetics (halothane, enflurane, isoflurane, diethyl ether, cyclopropane, nitrous oxide and chloroform) on the following processes in rat brain (hippocampus): 1) Ligand binding to glutamate binding sites on the NMDA receptor complex, including receptor density, affinity and kinetic constants using [3H]CGS 19755 as a specific probe. 2) Ligand binding to the glycine modulatory site, using [3H]CGS 19755 as a specific probe. 3) Glutamate, glycine, divalent cation and spermidine activation of NMDA receptor ion channels, using [3H]MK-801 as the probe. [3H]MK-801 binding is allosterically stimulated by glutamate, glycine and polyamines (such as spermidine) and inhibited by divalent cations (such as Zn2+). 4) NMDA-receptor mediated changes in calcium content of rat brain microvesicles due to flux through transmembrane channels and release from intracellular stores, using a fluorescent dye and 45Ca. The potency of the different anesthetics in affecting NMDA receptor binding functions will be compared to their 1) anaesthetic potency, 2) physiochemical properties, and 3) membrane-perturbing actions, in order to assess the relationship of these actions to the elaboration of the anesthetic state. This research will increase our understanding of volatile anesthetic action on signal transduction processes in the brain, and explore a mechanism that may contribute to the development of the anesthetic state.
