This application concerns the function of the outer plexiform layer after injury and during normal activity. Questions are addressed using freshly isolated photoreceptors and long term cultures of retinal neurons from adult retina. 1) Although adult salamander retinal neurons can form synapses in culture, it is not clear whether synapse formation is selective. Cells will be placed with appropriate and inappropriate partners by micromanipulation. The development of functional synapses will be monitored both anatomically and physiologically. By comparing with the known synaptology of the intact retina, the spectrum of connections possible during regeneration can be determined. 2) The photoreceptor synaptic terminal is capable of structural change. The initial stages of process outgrowth and synaptogenesis by salamander and primate rod and cone cells will be followed with time-lapse video microscopy whereas the movement of synaptic organelles will be examined in the confocal laser scanning and electron microscopes. Experiments will determine whether direct or transneuronal injury to the photoreceptors effects synaptic regeneration and what happens to the original synaptic organelles as new synapses are formed. 3) For photoreceptors, the rate of transmitter release by vesicle exocytosis in the dark and during the light response and the pathway of synaptic vesicle membrane recycling is not known. Rapid freezing of salamander and rabbit photoreceptors in conjunction with pulse labeling with electron dense tracers will allow quantification of vesicle release during synaptic activity and identification of recycling organelles. 4) Age-related retinal degeneration in old salamanders will be identified with optomotor reflex testing. Whether degenerating rod cells transport opsin to the outer segment and release transmitter in the dark will be examined with immunocytochemistry and synaptic activity dependent tracer uptake. In addition, the ability of aging photoreceptors to regenerate in culture, will be compared to regeneration by young cells. These experiments will increase our understanding of the potential for repair in the retina, answer general biological questions about secretion and cell organelle function, explore synaptic transmission by photoreceptors, and test photoreceptor activity during aging. In the long term, they may inform the procedures and choice of tissue for retinal transplantations as well as guide therapeutic techniques to stimulate endogenous repair of retinal injury or degeneration.
