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.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY003664-09
Application #
3258067
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1981-05-01
Project End
1992-04-30
Budget Start
1989-05-01
Budget End
1990-04-30
Support Year
9
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
Ruiz, M; Egal, H; Sarthy, V et al. (1994) Cloning, expression, and localization of a mouse retinal gamma-aminobutyric acid transporter. Invest Ophthalmol Vis Sci 35:4039-48
Sarthy, V (1993) Collagen IV mRNA expression during development of the mouse retina: an in situ hybridization study. Invest Ophthalmol Vis Sci 34:145-52
Burg, M G; Sarthy, P V; Koliantz, G et al. (1993) Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis. EMBO J 12:911-9
Sarthy, P V (1991) Histamine: a neurotransmitter candidate for Drosophila photoreceptors. J Neurochem 57:1757-68
Sarthy, P V; Fu, M (1990) Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization. J Cell Biol 110:2099-108
Sarthy, P V; Fu, M (1989) Transcriptional activation of an intermediate filament protein gene in mice with retinal dystrophy. DNA 8:437-46
Sarthy, P V; Fu, M (1989) Localization of L-glutamic acid decarboxylase mRNA in monkey and human retina by in situ hybridization. J Comp Neurol 288:691-7
Sarthy, P V; Fu, M (1989) Localization of L-glutamic acid decarboxylase mRNA in cat retinal horizontal cells by in situ hybridization. J Comp Neurol 288:593-600
Sarthy, P V; Fu, M; Huang, J (1989) Subcellular localization of an intermediate filament protein and its mRNA in glial cells. Mol Cell Biol 9:4556-9
Gaur, V P; Eldred, W; Possin, D E et al. (1989) A monoclonal antibody marker for the paraboloid region of cone photoreceptors in turtle retina. Cell Tissue Res 257:497-503

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