This study is directed at elucidating the morphological and physiological organization of the inner plexiform layer of the mammalian retina. It focuses on examining the ultrastructure of physiologically identified ganglion cells in the rabbit retina, and will help interpret our existing morphological data pertaining to the synaptic profiles of cone bipolar axonal terminals with amacrine and ganglion cells. Intracellular recordings are obtained under visual observations using infrared illumination and DIC optics on an isolated, superfused retina preparation. These recordings will determine: whether the cells respond to the onset or termination of light in a sustained or transient manner; the nature of their photoreceptor input; and whether the receptive field properties undergo any reorganization with changes in the adaptive state of the cells. Once physiologically identified, the cells are injected with Lucifer Yellow to verify their morphology with the light microscope; the Lucifer is photoconverted to an osmophilic precipitate, which can be visualized with the electron microscope; and serial micrographs of the labeled cell are used in computer graphics to reconstruct the synaptic connections of several major dendrites. Special attention will be focused on ascertaining a relationship between the amount of amacrine and cone bipolar cell input the ganglion cell receives with respect to its physiological responses to light. A new technique has been developed for the simultaneous, ultrastructural examination of two different morphologically and physiologically identified neurons to demonstrate that they form the pre- and postsynaptic processes of a synapse. This method employs the use of separate intracellular injections of HRP and a fluorescent dye (Lucifer Yellow) into different cells which have overlapping dendritic trees and are suspected of synapsing with each other. The tissue is sequentially reacted under two different conditions using two different chromogens: henzidine dihydrochloride is used to react the HRP, and diaminobenzidine is used to form the reaction product during the photoconversion of Lucifer. The fluorescent dye cannot be photoconverted in the presence of any chromogen unless it is maximally excited with its specific wavelength light. These two chromogens produce distinctly different appearing reaction products that can be differentiated at the light and electron microscope. This method will be used to determine whether the DAPI-accumulating, flat cone bipolar cells synapse with off-ganglion cells in sublamina alpha of the IPL, and if the starburst amacrine cells synapse with the on-off directionally selective (DS) ganglion cells. Variations in the synaptic profiles among amacrine, bipolar and DS ganglion cells, for which the preferred-null axis has been identified, will be examined to evaluate the morphological substrate for the physiological models of DS ganglion cells. Finally, by obtaining intracellular recordings from DAPI-accumulating cone bipolar cells, and injecting them with Lucifer to correlate their morphology with the previously examined cells, this study will attempt to provide conclusive evidence that the are off-bipolar cells.
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