Acetylcholine (ACh) affects the response properties of many retinal ganglion cells. The 9 alpha and 3 beta neuronal nicotinic ACh receptor (nAChRs) subunits can form many nAChR subtypes, each with a unique pharmacology and physiology. The proposed studies will test the hypotheses that 1) nAChR subtypes are differentially expressed in specific amacrine and ganglion cells types. 2) specific nAChR subtypes serve different functional roles in the retina, and these differences are reflected in their differential subcellular distribution. 1. To determine the nAChR subtypes expressed by ganglion cells and evaluate the spatial relationships of the nAChR-bearing cells to those of the starburst cells: Most ganglion cells in the retina are affected by cholinergic agents, but the nAChR subtypes that mediate these responses are unknown. Functional nAChRs expressed on ganglion cells will be studied with electrophysiological methods. The effects of subtype-specific agonists and antagonists on the light responses of physiologically characterized ganglion cells will be studied, and the effects of subtype-specific reagents on synaptically isolated cells' responses to cholinergic agonists will be evaluated. The cells will be filled with dye and the relationship of their dendrites to those of the starburst cells will be evaluated. 2. Antisera production: Antisera against synthetic peptides corresponding to unique sequences of mammalian alpha and beta subunits will be generated. These will be used for studies described in Specific Aims 3, 4, and 5. 3. To determine the subunit composition and abundance of nAChR subtypes: Our data suggests at least 3 nAChR subtypes are expressed in the rabbit retina. RT-PCR analysis will be used to determine what subunits are expressed in the retina. Immune precipitation experiments using existing antibodies and others in development will be used to determine the subunit composition of rabbit retinal nAChRs and to quantify their relative abundance. 4. To identify the nAChR-expressing cells in the mammalian retina: Antibodies and antisera against alpha and beta subunits will be used to determine the patterns of expression in rabbit retina, nAChR-expressing cells will be identified based upon their content of neurotransmitters, enzymes, or other molecular markers as revealed by double label studies. 5. To determine the subcellular localization of nAChRs in the IPL: Our data suggest that most of the alphaBetagt-insensitive-beta2alpha3 nAChRs are not localized to synapses. Alpha7 nAChRs in the brain are reported to be pre- or perisynaptic and functional alpha7 nAChRs are expressed on ganglion cells. Electron microscope analysis of the distribution of these receptors in the IPL will be conducted to determine if the nAChRs are associated with synapses only in the strata that contain the cholinergic cell dendrites.
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