This is a request for an ADAMHA Research Scientist Award. The application requests funds to allow me to pursue the acquisition of techniques of molecular biology and neuroscience and their application to 2 projects. The first project will examine by solution hybridization, Northern blot analysis, in situ hybridization and immunocytochemical techniques the mRNA and peptide levels for proenkephalin (Penk) prodynorphin NMDA receptor, nitric oxide synthase, c-fos and tyrosine hydroxylase in 15 select areas that are associated with opioid analgesia, tolerance and dependence. These studies will be conducted in animals made tolerant and/or dependent with mu, kappa and delta opioids and in the presence and absence of drugs (receptor antagonists and nitric oxide synthase inhibitors) which attenuate or prevent the development of opioid tolerance and dependence. In addition, a subtractive hybridization approach will be used to clone and identify proteins altered by opioid tolerance and dependence. The second project will define the factors including membrane depolarizing,second messenger systems and glucocorticoid that regulate the hamster, rat and human Penk genes. Glucocorticoid response elements in the hamster, rat and human Penk genes will be identified by cloning selected DNA fragments. These projects require the acquisition and application of a number of molecular and neuroscience techniques including in situ hybridization, subtractive hybridization, tissue culture, fusion gene construction, gene transfer, gel-shift mobility assays and transgenic analysis. The acquisition of these techniques will result from collaborations with scientists with expertise in each of these areas who have agreed to support the acquisition and transfer of these techniques to my laboratory. This RSA award will allow me the opportunity to reduce my teaching and administrative responsibilities and increase the time I can devote to enhancing my scientific skills.
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