This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this project is to determine the morphology, physiology, and central connections of ganglion cell types using a new retrograde tracing method that we call 'retrograde photodynamics'. Over the last two years we used rhodamine-dextrans to label macaque monkey ganglion cells from tracer injections in the major retinal targets: the superior colliculus, pretectum, and LGN. After retrograde transport, the tracer was sequestered into organelle-like structures within ganglion cell bodies and proximal dendrites. This particulate labeling alone did not allow unambiguous targeting of specific cell types in vitro. We discovered that when labeled cells were observed microscopically under epifluorescent illumination, the fluorescing organelles appeared to burst, creating a fireworks-like display in the cytoplasm and the liberated fluorophore rapidly diffused throughout the dendritic tree. At the same time, a large increase in fluorescence intensity within the cytoplasm gave rise to a bright and complete intracellular dye stain. Photostained cells remained anatomically and physiologically viable; we could target morphologically distinct types in vitro for intracellular recording and analysis of receptive field properties. Further, by employing the biotinylated form of rhodamine dextran, it was possible to use horseradish peroxidase (HRP) histochemistry after tissue fixation to permanently recover the detailed morphology of large numbers of cells for anatomical analysis. This method enabled us to characterize several new ganglion cell populations that project in the primary visual pathway to the LGN. Some of these cell groups show novel color-opponent properties and will be a continuing focus of new research projects. One of these groups, the giant monostratified cells, are uniquely photosensitive and form the basis for another project in the lab. We are also interested in further immunohistochemical studies of melanopsin-reactive cells in the retina as well as their central terminals, and have enlisted a collaborator in Denmark to this end.
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