The demonstration that anesthetics affect the protein firefly luciferase has challenged the prominent theoretical position that anesthetics act by disrupting lipid membranes. These newer data suggest that hydrophobic domains of neural membrane associated proteins are more likely to be involved in anesthetic actions than lipid solubility. In this regard, since GABA/A receptors are made up of differing protein subunits with varying characteristics (i.e., from differing receptor subtypes) and hydropathy plots, it would seem reasonable that some GABA/A receptors formed by these proteins would be altered differentially by anesthetics. Preliminary evidence indicates that low concentrations of isoflurane affect some, but not all, responses to GABA, providing a reasonable explanation for the differential effects of this anesthetic on CNA functions. We will test the hypothesis that isoflurane has this differential action on GABA responses by affecting specific GABA/A receptor subtypes. The first experimental series will determine if this differential action of isoflurane on responses to GABA is seen on neurons in the medial and lateral septum at several concentrations of isoflurane administered systemically. Additionally, it will be determined whether Type I, Type II, or non-benzodiazepine GABA/A receptors are equally sensitive to isoflurane in vivo. Subsequently, the action of isoflurane will be tested on individual dissociated neurons from the septum to identify neurons sensitive and insensitive to isoflurane enhancement or inhibition of GABA responses. The GABA/A receptor subunits within these dissociated neurons sensitive and insensitive to these actions of isoflurane will be identified using RT-PCR of the mRNA for the subunits. Once the GABA/A receptor subunits associated with these actions of isoflurane are identified, these GABA/A receptor subunits forming the receptor will be mapped in brain using immunohistochemistry and in situ hybridization. At brain sites where these identified subunits are found, electrophysiological studies will be performed in vivo and in vitro to confirm the hypothesis that the subunits within the GABA/A receptor complex dictate the action of isoflurane on responses to GABA. This new information, using the GABA/A receptor to illustrate the selectivity of isoflurane for specific receptor subtypes, is expected to provide an explanation for the ability of anesthetics to depress some, but not all, physiological functions during induction and surgical anesthesia, lending a new perspective and understanding of the manner by which anesthetics can differentially affect CNS function.
