The purpose of this core is to provide and maintain equipment, reagents, and expertise necessary to examine the localization of receptors for excitatory neurotransmitters at the cellular and subcellular level. Given the primary sequence information now available for many subtypes of excitatory transmitter receptors, straight-forward techniques are available to obtain highly selective labels for these molecules that can be applied in anatomical investigations. The emphasis of this facility will be on the development of agents and methods for immunocytochemical studies. Polyclonal antibodies that label specific receptors for excitatory transmitters will be generated using the anti-peptide antibody approach. Some anti-receptor antibodies are presently available through collaborators; these pertain to acetylcholine and glutamate receptor subtypes that were cloned several years ago. The applicants have successfully raised and purified antibodies against a newly described glutamate receptor (mGluR5) and are currently purifying antibodies we have raised against a recently cloned NMDA receptor (NR1). One of our mGluR5 antibodies appears to be highly specific for this receptor subtype. We have successfully applied this antibody for light microscopic immunocytochemical localization studies and are currently developing methods for immunoelectron microscopic application of this antibody. We have made arrangements to receive from Dr. Shigetada Nakanishi cDNA clones for various glutamate receptor subtypes which we will use to develop cell lines that express these receptors. These cell lines will provide an additional valuable means of confirming the receptor subtype specificity of the antibodies we generate. Image analysis hardware, as well as the equipment for light and electron microscopy are already available and are not requested for the core. The immunological probes and methods developed by this core will be a valuable asset for several of the projects in this research program.
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