Steroids are effective general anesthetics at brain levels far below those observed for barbiturate or inhalational anesthetics. And closely related steroids are present at very different brain levels at the same behavioral endpoint of anesthesia. These finding contrast with the equivalent membrane concentrations predicted from classical correlations between anesthetic potencies and oil/water partition coefficients. In this grant application experiments are proposed to define the molecular target for steroid anesthetics. It is suggested that the target could be a receptor molecule able to distinguish among closely related ligands. Studies in vivo and in vitro will test this hypothesis. The loss of the righting reflex (LRR) is our behavioral endpoint for the anesthetized state. Mice (and in separate experiments, tadpoles) are anesthetized with 3H steroids and sacrificed at the LRR. Steroids are extracted from the brain and chromatographed (HPLC or TLC), and quantitated by scintillation spectrometry or u.v. absorbance. Potencies of related steroids are used to characterize interactions with the putative receptor. Inhibitors and partial agonists are identified. Interactions between steroids and other general anesthetics are assessed. The criteria for the existence of a receptor are experimentally evaluated in vitro with homogenates from brain. These include saturability, reversibility, and a correspondence of in vivo and in vitro activities comparing rank order potencies, binding affinities, and agonist and antagonist actions. The broadly hypothesized possibility that steroids act at the GABA A receptor will be evaluated by in vitro studies of ligand binding (e.g. GABA agonists, benzodiazepines) and ion (36Cl-) flux. a steroid photoaffinity ligand will be synthesized and used in covalent binding studies to identify, purify and localize a receptor molecule. These are important steps to understanding of the mechanisms that underlie general anesthesia.
