? ? This application is a competitive renewal of a currently funded K02 award and is focused on advancing the PI's ability to investigate the actions of ethanol on brain ion channels. The PI has conducted research into the effects of alcohol of the NMDA receptor and is currently funded by NIAAA through an R01 to continue this work. The PI has utilized the currently funded K02 award period to obtain training in two-electrode voltage-clamp, whole-cell patch-clamp eletrophysiology and molecular biology. He has used these techniques to make important discoveries with regard to the molecular sites of alcohol action of the NMDA receptor. Results from experiments utilizing transfection of oocytes and HEK293 cells with wild-type and mutant NMDA receptors has led to a better understanding of how subunit assembly, intracellular signaling processes and transmembrane domains modulate the sensitivity of this receptor to alcohol. In this application, the PI will extend the relevance of these findings by learning techniques used to express mutant NMDA receptor subunits with altered ethanol sensitivity in brain neurons and to assess specific areas of molecular biology, electrophysiology, and animal behavior. Specifically, the PI will: 1) learn techniquesneeded to generate and use viral-based gene vectors to deliver NMDA receptor subunits with altered ethanol sensitivity into neurons grown in culture and in vivo; 2) learn methods used to generate genetically modified animals that express mutant NMDA receptor subunits with altered ethanol sensitivity; 3) learn methods to prepare and record ion channel currents from brain slices isolated from animals expressing mutant NMDA receptors with altered ethanol sensitivity and; 4) learn techniques needed to assess the neurobehavioral actions of alcohol in animals expressing mutant NMDA receptors with altered ethanol sensitivity. This training will be obtained during a series of sessions supervised by a variety of consultants with demonstrated expertise in the techniques. The successful completion of this training plan will substantially enhance the PI's research expertise and his ability to fulfill the goals of his research.
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