This research proposal is concerned with a study of synaptic mechanisms in the outer plexiform layer of the vertebrate retina. Pre-and postsynaptic function will be examined separately by using freshly isolated retinal neurons and synape formation between mature neurons will be examined in long term cultures of dissociated retinal cells. 2) Presynaptic transmitter release by exocytosis can be quantified with the rapid freezing technique in amphibian and mammalian rod cells. This approach will yield information on the rate of synaptic vesicle release in the dark and during the light response. Synaptic vesicle membrane recycling will be followed by pulse labeling with ultrastructural lables and rapid freezing. By combining these techniques it will be possible to identify the organelles which retrieve synaptic membrane, regenerate synaptic vesicles, and transport membrane to the cell body. 2) the role of Ca++ in synaptic activity and phototransduction will be explored by identifying the Ca++-sequestrating organelles of active photoreceptors with rapid freezing and freeze substitution. 3) Cholinergic receptors will be localized on first and second order neurons with radiolabeled ligands and light and electron micorscopic autoradiography. Using competitive ligands it will be possible to selectively visualize the binding sites of muscarinic subtypes and determine their binding affinities both for specific cell types and for parts of neurons. and 4) The fate of denervated presynaptic active zones and the specificity of newly formed synapses will be determined with morphological and patch-clamp examination of selected pairs of retinal neurons maintained in vitro. These experiments on synaptic function answer general biological questions concerning secretion and cytoplasmic organelle function; they explore how visual information is communicated by photoreceptors to second order neurons; they examine the possible cholinergic interactions in the outer plexiform layer; and they test the ability of adult neurons to regenrate. In the long term, it is hoped that they will provide a basis for the use of cell culture techniques in studies of photoreceptor cell biology and synaptic interactions. The continued analysis of the normal function of visual cells is essential in order to understand the alterations which occur in retinal and choroidal disease processes. For the protoreceptor, this may be especially important since rod/cone degeneration is a primary defect in a number of retinal disorders whose etiology is essentially unknown.
