Although volatile anesthetics have been used in surgical procedures for over 150 years, their mechanism of action remains an enigma. This research proposal aims to identify the mechanism of action of prototypic volatile anesthetics using a murine genetic model. The proposal details an approach not previously applied to investigations of mechanisms of volatile anesthetic action. Two methods identified a region on mouse chromosome 7 which influences volatile anesthetic response 1) a computational method correlating the phenotypic response to volatile anesthetics among 13 inbred mouse strains with the pattern of single nucleotide polymorphisms (SNPs) among these strains; and 2) analysis of backcross progeny derived from 2 parental strains with different anesthetic (isoflurane) responses. Eight candidate genes coding for ion channels expressed in the spinal cord, which is the neural substrate of volatile anesthetic-induced immobility, were identified within the experimentally and computationally identified region on mouse chromosome 7. This proposal has three specific aims.
The first aim characterizes the 8 candidate ion channels within the identified region on chromosome 7. Ion channels isolated from two mouse strains exhibiting high and low responses to anesthetic agents will be evaluated for qualitative differences in electrophysiologic experiments on expressed channels in vitro, and for quantitative differences by evaluation of RNA expression in the spinal cord in vivo. The goal is to identify ion channels that display strain differences at a molecular level that plausibly account for the difference in anesthetic response between strains.
The second aim i dentifies other genomic intervals that contain genes influencing anesthetic potency. Because response to volatile anesthetics is not inherited in simple Mendelian fashion, in addition to the region identified on chromosome 7, other genomic intervals must influence this response. Backcross progeny from high and low anesthetic response strains will be generated and evaluated using a SNP-based genome scan to identify these regions.
The third aim produces congenic mice in which the identified chromosome 7 segment from mice with high responses to anesthetic is transferred onto the genetic background of the low responder strain, in order to quantitatively assess the effect of this region (and hence the genes in this region) on anesthetic potency.
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