Acetylcholine (ACh) is used as a transmitter in the vertebrate retina. Recently, alpha and beta subunits of neuronal nicotinic acetylcholine receptors (nAChRs) and alpha subunits of alpha-bungarotoxin-sensitive nACbRs (alphaBgtAChRs) have been purified, their cDNAs cloned, and antibodies raised against them. These studies have shown that there are many different alpha and beta subunits that can combine to form a large number of receptor subtypes that are physiologically distinct. Thus, the effects of ACh depends upon which receptor subtypes are present at specific synapses. The proposed research represents an extension of previous efforts to determine 1) the pattern of subunit expression in the retina, 2) the identity of receptor-containing cells and the spatial relationship of their dendrites and the processes of other retinal cells, 3) the amounts of the various subunits in the retina, and 4) which subunits are combined in the same receptor complexes. 1. Immunohistochemical localization of nAChR and alphaBgtAChR subunits in the retina. Structural and ligand-binding subunit-specific antibodies will be used at the light microscope level to determine the distribution of subunits in somata and neuropil. Double label studies will be used to identify somata and processes that contain multiple nAChR and/or alphaBgtAChR subunits. Electron microscopy will be used to define the relationship of subunit immunoreactivity to cellular and synaptic structures. 2. Identification of the receptor-containing cells, and the spatial relationships between their dendrites and the processes of other retinal neurons. Double label studies that rely on subunit specific antibodies and antibodies against either protein kinase C or neurotransmitters (or their synthesizing enzymes) known to be associated with specific cell populations will be used to classify receptor-bearing amacrine, bipolar, and ganglion cells. Other double label studies will examine the association of receptor-containing processes with those of transmitter- containing cells. 3. Measurements of the relative amounts of each subunit in the retina and determination of which alpha subunits are found in the same receptor complex. Immune precipitation experiments using subunit-specific monoclonal antibodies will be used to determine the relative amounts of the different nAChR or alphaBgtAChR receptor subunits in mammalian retina. Other immune precipitation studies will determine what nAChR alpha subunits are found in association with each other and whether any of these subunits are found in """"""""hybrid"""""""" complexes with alpha subunits of alphaBgtAChRs.
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