The long-term objective of the proposed research is to further understand the cellular mechanisms of inhaled anesthetic action. The proposed research focuses on the involvement of GABAA receptors containing the alpha5 subunit in the central depressant effects of isoflurane and other inhaled anesthetic agents. Dr. Topf is a clinician-scientist, seeking further training in neuropharmacological and genetic techniques. He will conduct the proposed research in Dr. Neil Harrison's laboratory at Weill Medical College of Cornell University. Dr. Harrison is a neuroscientist who is a world authority on the pharmacology of the GABAA receptor and a leader in the field of molecular mechanisms of general anesthetics. The alpha5 subunit of the GABAA receptor is expressed at high levels in the hippocampus, a structure known to be involved in learning and memory; we therefore hypothesize that alpha5-containing GABAA receptors could play an important role in the clinically relevant amnestic effects of volatile anesthetics. To further investigate the pharmacology of volatile anesthetics at GABAA receptors containing the alpha5 subunit we will conduct a series of pharmacological experiments with recombinant wild type and mutant receptors in vitro. We will also clone fragments of the mouse genomic DNA encoding the alpha5 subunit, enabling the future production of a 'knock-in' mouse expressing a mutated a5 subunit with altered volatile anesthetic pharmacology. The detailed aims are: 1. Express wild type and mutant GABAA receptors containing the alpha5 subunit (alpha5beta3gamma2s) in HEK 293 cells and study the effects of isoflurane and halothane on these receptors. 2. Investigate the kinetics of receptor activation/ deactivation of these wild type and mutant receptors and the effects of inhaled anesthetics. 3. Clone the mouse genomic DNA encoding exon 8 of the alpha5 subunit and create a targeting construct incorporating specific mutations in the alpha5 subunit that allow for normal responsiveness to GABA, but render the receptor insensitive to isoflurane. The results of these studies will provide new insights into the molecular mechanisms of volatile anesthetics at the a5 subunit of the GABAA receptor. This work will provide the foundation for further investigations of the role of alpha5-containing GABAA receptors in the central effects of volatile anesthetics such as isoflurane. It will provide the necessary training and experience for Dr. Topf to establish himself as an independent investigator.
| Perrier, Anselme L; Tabar, Viviane; Barberi, Tiziano et al. (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101:12543-8 |
