Our goals are to examine the development and reconstruction of retinal circuits 1) that must form after retinal transplantation if some vision is to be restored, 2) that occur in the degenerate retina as the retina is remodeled, and 3) that function in the normal retina. We propose to do this by combining the technologies of optical tweezers with microelectrode arrays on glass chips. Chips will be designed to have non-adherent and adherent zones for cell movement and cell culture respectively. Using the optical tweezers, isolated adult retinal cells that are identified by their distinctive morphologies will be micromanipulated onto electrodes and allowed to form cell-cell contacts. Synaptic development will be monitored by immunocytochemistry and synaptic vesicle recycling dyes. Synaptic activity will be tested by electrode stimulation and imaging of non- toxic voltage-sensitive dyes for bipolar and some amacrine cells, and by recording electrodes for the spiking and action potentials of amacrine and ganglion cells. The use of the tweezers allows precise control over the circuits that are built. The ability to design and then functionally test specific nerve cell interactions over time will provide a system with an unprecedented level of experimental control.
