The cDNAs encoding many of the neurotransmitter receptors have been cloned recently. Consequently, we can now begin to identify both the retinal cells that contain a transmitter and those that bear the receptors for the transmitter. A fundamental observation is that the ionotropic-type receptors are quite diverse; for each neurotransmitter there are many different receptor subunits and an even greater number of potential subunit complexes. It is possible that for each transmitter used in the retina there are many different receptor-subtype specific synapses. The goal of the proposed work is to explore this heterogeneity by defining how the Kainate/AMPA-type glutamate receptor subunits are expressed and combined by the cells of the retina. Glutamate is a neurotransmitter fundamental to retinal function, and in situ hybridization experiments in our laboratory have revealed that the mRNAs encoding the seven cloned subunits (GluR1 through GluR7) are expressed in the retina. 1). Tools: the generation of a) glutamate receptor subunit-specific antisera and b) subunit-bearing cells: Subunit-specific antibodies must be created to study the actual receptor subunit proteins. To do this, the most dissimilar portions of each of the cDNAs encoding GluR1 through GluR7 will be subcloned into plasmids for bacterial overexpression. The resulting fusion proteins will be used as antigens to immunize rabbits and goats. Western blots of the fusion proteins will be used to identify immune responses. Kidney cells will be transfected with each of the receptor subunits to provide independent verification and validation of the subunit- specificity of the antisera in each of the following experiments. 2). Anatomy: the immunohistochemical localization of the glutamate receptor subunits in the retina: Subunit-specific antisera will be used to immunohistochemically localize the receptor subunits. Light and electron microscopy will be employed to define the cellular localization of the receptor immunoreactivity and to identify as best as is possible the receptor-bearing cells. Double labeling strategies will be used to determine which sets of receptor subunits are co-expressed. 3). Chemistry: the characterization of the receptor subunit proteins and the complexes they form: First, Western blots will be used to analyze the receptor subunit proteins in the crude membrane fraction of retina. Then, the rabbit antisera will be used to immune precipitate solubilized receptor complexes from the retina. These will then be fractionated on gels, blotted and probed with the goat subunit-specific antisera. The goal will be to define the sets of subunits that associate with one another.
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