The studies described in this proposal are designed to respond to the RFP for the development of antibodies to receptors and signal transduction macromolecules important in the expression of the effects of ethanol on brain function and behavior. We plan to generate new antibodies to various NMDA receptor subunits and to specific domains of these proteins. The antibodies we plan to raise are targeted to regions of the proteins that represent key regulatory sites for the function of the proteins. Numerous studies have confirmed that a prevalent site of ethanol action in the central nervous system is at the glutamate receptors, in particular the NMDA receptors. Although there is remarkable agreement on the interference by ethanol with normal glutamatergic neurotransmission in brain as a result of the inhibition of NMDA receptor-ion channels, there are still several issues that need to be examined with the use of new molecular probes. The antibodies we plan to raise should help many investigators interested in probing some of these issues.
The specific aims for this project are:
Specific Aim #1. The subcloning and expression of antigenic proteins or the synthesis of peptides based on the structure of the NMDAR1/NMDAR2 receptor proteins and of the glutamate, CPP, and glycine binding protein subunits of a receptor-like complex for the purpose of raising polyclonal and monoclonal antibodies.
Specific Aim #2. The characterization of the immune reactivity of the antibodies raised by means of immunochemical assays, Western blots, immunoextraction procedures, and immunocytochemical tracing of the expression of the proteins in brain.
Specific Aim #3. The characterization of the activity of select antibodies as inhibitors of NMDA-induced Ca2+ influx into neurons and as probes of the altered expression specific subunit isoforms in the brains of animals exposed to chronic ethanol treatment or in primary neuronal cultures treated in vitro with ethanol.
Specific Aim #4. The full documentation of the properties of each antibody, either polyclonal or monoclonal, generated and the production, storage and distribution to other scientists of these antibodies.
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