One concept to explain the etiology of ethanol-related neurological disorders is that ethanol alters the normal function of fast neurotransmission receptor systems in the brain. These receptors include the neuronal nicotine-activated receptors (nAChR) and glutamate-activated receptors (GluR). Understanding the mechanism(s) of ethanol's specific effects on these receptors is crucial to interrupting the habituating behavior and enhanced susceptibility to neurotoxicity that correlates with abuse of this compound. The intent of this RFA is to generate and distribute antibodies to proteins targeted by alcohol. This is the theme of our proposal and the interactive proposal submitted by Dr. Ronald Lukas of the Barrow Institute. We propose two Specific Aims. First, we will generate polyclonal and monoclonal antibodies to nAChR and GluR subunits and define their subunit specificity and their most useful application for immuno-detection of these proteins. Bacterially-produced fusion proteins or fusion-peptides will be used as immunogens, and the resulting antibodies will be screened and tested using a combination of Western blot analysis, immunoprecipitation, and immunohistochemistry. Major immunogenic regions will be defined by epitope mapping of specific immunoreactivities from the polyclonal antisera and this information used as a """"""""road map"""""""" to generate appropriate mouse monoclonal antibodies in amounts sufficient for distribution to other investigators. Second, we will use these antibodies for immunohistochemical analyses. Specifically, our experiments will test the hypothesis: Ethanol sensitive mice (LS or 'long-sleep') differ relative to ethanol insensitive mice (SS or 'short-sleep') in the expression and/or distribution of GluR and/or nAChR subunits normally or subsequent to chronic ethanol ingestion.
