The research goal is a novel application of multiphoton laser scanning microscopy to determine neural coding mechanisms of diverse cell types and local circuits in the vertebrate retina that have been previously inaccessible by current experimental methods. Our specific request is to acquire a scanning laser microscope with specialized features that uniquely permit optical recording of neural signals from retinal neurons while simultaneously stimulating the retina with complex visual stimuli. The requested instrument is a BioRad 2000 MP two-photon scanning laser microscope coupled to a femtosecond laser light source and an open optical architecture that allows optical recording of calcium signals evoked by light stimulation of the intact retina maintained in vitro. The critical application of this instrument to vision research is dependent on the confinement of fluorescence activation to a spatially restricted femtoliter-sized volume at infrared wavelength with insufficient energy to directly excite the light sensitive retinal photoreceptors. The requested instrument is a newly developed tool that is specialized for retinal research and will be used almost exclusively by three major investigators whose currently funded specific aims are focused on mechanisms of signal processing by identified retinal neurons. Each investigator will use the instrument to determine the link between calcium signals in morphologically identified dendritic processes and specific features of retinal and visual function. Detwiler proposes to further investigate the basic properties of the light-evoked calcium signals. This will include understanding their dependence on visual stimuli, their biophysical properties and the molecular mechanisms responsible for their generation. Rieke proposes to determine how mechanisms in the synaptic processes of rods, bipolar cells and amacrine cells transform the rod-mediated light response. Dacey proposes to determine how a color code in the primate retina is computed at the dendritic tree of newly identified retinal cell types. Because of the specialized application of this research to visual neuroscience the instrument will be linked to existing equipment needed for physiological recording and light stimulation. The instrument will be heavily used by each of the three investigators since the microscope would be engaged on a daily basis in experiments that will run for several hours.
