CHOLINOCEPTIVE NEURONS IN THE RETINA

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Acetylcholine (ACh) is a transmitter in the vertebrate retina that affects the response properties of many ganglion cells. Eight alpha and three beta subunits of neuronal nicotinic acetylcholine receptors (nAChRs) have been cloned, and antibodies have been raised against them. These subunits can combine to form a large number of nAChR subtypes, each characterized by a unique subunit composition and physiological and pharmacological profile. The specific aims are based on the hypothesis that 1) different retinal cell types express different combinations of nAChR subtypes, and that 2) different nAChR subtypes serve different functional roles in the retina. 1. Antisera production: The production of antisera against synthetic peptides corresponding to unique sequences of chick and mammalian alpha and beta subunits will be continued. These will then be used for use in the studies described in Specific Aims 2, 3, and 5. 2. Characterization of the nAChR-expressing cells in the avian and mammalian retina: Antibodies and antisera against alpha and beta subunits will be used to determine the nAChR expression pattern in the chick, rat, and rabbit retina. nAChr-expressing cells will be identified based upon their content of neurotransmitters, enzymes, or other molecular markers as revealed by conventional double label studies. The spatial relationships between the dendrites of the nAChR-expressing cells and the processes of other neurons will be studied in the same fashion. 3. Determination of the subunit composition and abundance of retinal nAChR subtypes: Immune precipitation experiments using the same antibodies and antisera as above will be used to determine the subunit composition of chick and rabbit retinal nAChR and to quantify their relative abundance. 4. Characterization of subpopulations of cholinergic amacrine cells: The cholinergic amacrine cells of the rabbit retina comprise at least two subpopulations based upon nAChR and GABA expression. These subpopulations will be characterized in terms of their density, distribution, and the spatial relationships between the dendrites of these cells and those of other retinal neurons. 5. Cholinergic circuitry in the developing mammalian retina: Subunit specific antibodies and antisera will be used in conjunction with commercially available antisera to determine the pattern of choline acetyltransferase, GABA, and nAChR expression in retinas from postnatal ferrets of known ages.