This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is to determine the morphology and physiology and central connections of ganglion cell types using a new retrograde tracing method that we have called 'retrograde photodynamics'. We have used rhodamine-dextrans to retrogradely label macaque monkey ganglion cells from tracer injections in the major retinal targets: the superior colliculus, pretectum, and LGN. As expected after retrograde transport, the tracer is sequestered in organelle-like structures within ganglion cell bodies and proximal dendrites. This particulate labeling alone does not allow unambiguous targeting of specific cell types in vitro. However when labeled cells were observed microscopically under epifluorescent illumination, the glowing organelles seem to burst?creating a fireworks-like display in the cytoplasm?and the liberated fluorophore rapidly diffuses throughout the dendritic tree. At the same time, a large increase in fluorescence intensity within the cytoplasm gives rise to a bright and complete intracellular dye stain. Photostained cells remain anatomically and physiologically viable;we 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 is 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 enables us to rapidly 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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