This project is designed to uncover basic patterns of bipolar cell connectivity in the inner nuclear layer of the retina. The output of cones quickly diverges into many parallel pathways initiated at the cone to bipolar cell synapses. The properties of these channels are defined by the morphology and pharmacology of the bipolar cells, t;he spatial extent and stratification patterns of their dendrites, their pattern of cone contacts, and their degree of interconnectedness. Yet studies directly concerned with cone bipolar cells have been rare in mammalian retina, save for a few Golgi and electron microscopy studies and a very few electrophysiological recordings. This project aims to study: The morphology of bipolar cells. The PI will label bipolar cells with aDAPI, then stain them intracellularly with Lucifer Yellow. Their morphology and stratification patterns will be determined. Bipolar cell density, size and coverage factor will have measured across the entire retina. Nearest neighbors of a given type will be stained to show patterns of contact and local coverage. DAPI fluorescence will aid in clarifying the number of distinct bipolar cell types in the rabbit retina. Comparison will be made of individual cell types stained by the Golgi method, by antibodies selective for subtypes of bipolar cell, by dye injection following DAPI labeling, and by dye coupling via AII amacrine cells. Cone-to-bipolar cell contacts. Staining of the cone mosaic with lectins and anti-blue opsin will determine the convergence, divergence and cone specificity of the various bipolar cell types. These measures show how spatial and chromatic information is conveyed through the channels associated with the individuals bipolar cell types. Examination of the contacts made by bipolar cell dendrites with cone pedicles will be made with electron microcopy. Dye coupling in bipolar cells. The small tracer Neurobiotin will be injected into the various types of bipolar cell. Coupling to AII amacrine cells or other bipolar cells will be observed. The rate of Neurobiotin spread in bipolar cells can be compared to that in other cell types to quantify relative extent of dye coupling. This will assess the relative significance of the various gap junction in terms of pooled electrical signals and other small cytoplasmic molecules. The degree to which this spatial pooling contributes to mechanisms of adaptation and acuity can be inferred. Pharmacological manipulation of dye spread will indicate the extent to which light adaptation regulates coupling between bipolar cells and between bipolar cells and AII amacrine cells.
