The synaptic organization and physiological responses of the different classes of retinal neurons are fairly well understood. However, the neurotransmitters used by most of these neurons remain to be identified. In particular, the transmitters for the photoreceptor, bipolar and ganglion cells are yet to be established. A direct approach to this problem is to obtain homogeneous populations of the different retinal neurons and examine their neurochemical properties. Here, we propose methods for selectively marking the ganglion and bipolar cells with fluorescent dyes, and separating the labeled cells from nonlabeled cells using a Fluorescence-Activated Cell Sorter. Ganglion cells will be labeled by retrograde transport of the fluorescent dye, True Blue, injected into the optic chiasm. The bipolar cells will be marked by incubation of retinas with Lucifer Yellow CH in the absence of Ca++. The labeled cells will be dissociated after proteolytic treatment, and separated using the cell sorter. For obtaining enriched rod photoreceptor fractions, dissociated retinal cells will be first reacted with the rod-specific murine monoclonal antibody, Ret-P1, and then with magnetic beads coated with anti-mouse rabbit antibody. The bound cells are separated by Magnetophoresis. These preparations will be used to study the biosynthesis, uptake, release and metabolism of putative transmitters, and to localize transmitter receptor sites. In order to facilitate the separation and characterization of retinal neurons, we plan to obtain murine hybridomas which produce monoclonal antibodies to the different neurons in the rat retina. Finally, the development of GABAergic and Cholinergic neurons, and their interactions with other transmitters, will be studied in cell cultures of embryonic rat retina maintained in vitro. The identification and characterization of neurotransmitters is an important step in the treatment of retinal diseases, since such knowledge should provide a pharmacological basis for the design and testing of new drugs.
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