To help define the physiological targets of general anesthetics, we are studying mutants of Drosophila melanogaster that display an altered response to these agents. Since not every mutation that influences anesthetic effects on complex behaviors like standing and walking will do so by altering a direct target of the drug, we supplement such behavioral assays with electrophysiological monitoring of a reflex escape pathway. Our previous work has shown that the brain portion of this reflex but not the motor output portion contains at least one anesthetic- sensitive target. To see how strongly this target is influenced by changes in gross brain anatomy, we have examined the escape response pathway in several mutant lines known to have alterations in this anatomy. To accommodate our previous finding that anesthesia sensitivity is strongly influenced by genetic background; we have only used a set of mutations that have been made congenic with the Canton-S wild-type strain. We find that none of these mutations, which introduce mild to severe alterations in the central brain and mushroom body of the fly, interfere with the normal functioning of the escape response pathway; this confirms our surmise that the brain portion of the pathway is limited to the optic lobe. Moreover, most of these mutations fail to alter the anesthetic sensitivity of the response, permitting us to conclude that anesthetics work locally and not via widespread alterations of brain function. Only one of the loci tested, mushroom body defect (mud) changes the sensitivity of the fly in this assay. Because the mutant effect persists under conditions in which brain anatomy is virtually normal, we believe the mutation has a subtle affect on physiology of the adult fly. The mud gene maps to the same region of the X chromosome of Drosophila to which we have previously mapped another set of mutations, har, that influence anesthesia sensitivity. Although har mutants and mud mutants complement each other, their close proximity suggests that this segment of the X chromosome will be of high interest for understanding effects of anesthetics. Accordingly, we have begun a molecular genetic evaluation of this region. Within a 60kb interval that contains har and mud mutations, we have mapped more than 15 transcripts and have assessed their status in the mutants. We have also used Southern blotting and SSCP techniques to search, respectively, for gross rearrangements and subtle alterations of the genome associated with these mutations. While most of these techniques have not been revealing, our SSCP analysis leads us to believe that the mud gene is located at the centromere-proximal end of the 60kb window and encodes a large, novel protein with a high propensity to form many segments of coiled-coil structure.
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