Detailed studies of differentiated central neurons of the mammalian visual system would be aided by examining the membrane properties of an identified cell in a controlled in vitro environment. This approach requires that cells be isolated, unequivocally identified, and cultured. Cultures of solitary rat retinal ganglion cells, identified with fluorescent probes, are being developed in this laboratory. Since ganglion cells are the only retinal neurons that project to other areas of the central nervous system, they can be labelled by retrograde transport of fluorescent dyes injected into their projection sites, such as the superior colliculus and lateral geniculate nucleus. To confirm the identify of retrogradely labelled cells, a monoclonal antibody, that among retinal cells is specific for the ganglion cells, can also be used as a marker with immunofluorescent techniques. Following dissociation of the retina with papain and trituration, the labelled ganglion cells are placed in culture.
The specific aims of this proposal are-- (i) To characterize the various ionic currents of the cell membrane of solitary retinal ganglion cells. Conventional intracellular and patch-clamp electrodes will be used to record voltage as well as whole-cell and single-channel currents. Each current will be isolated using a combination of voltage-clamp, pharmacological agents, and ionic substitution. (ii) To test the effects of putative neurotransmitters and modulators on these currents. These substances include acetylcholine, GABA, glutamate, glycine, indoleamines, dopamine, and many peptides such as substance P, somatostatin, thyrotropin releasing hormone (TRH), and enkephalin. The long-term goal of this project is to further the understanding of intercellular communication between central neurons in the mammalian visual system.
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