DEVELOPMENT OF FEMTOSECOND SCANNING-LASER MICROSCOPY

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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 directly measure synaptic interactions on the dendritic trees of identified color opponent ganglion cells and to test the hypothesis that color opponency is determined by selective connections between cone-signal pathways and ganglion cell dendrites. To achieve this aim we were awarded an NIH shared instrumentation grant in 2001 to acquire a femtosecond, 2-photon scanning laser microscope for measuring light evoked calcium signals in the dendrites of retinal neurons. Over the last two years this facility was custom built, modeled after the system originally developed by Denk and Detwiler at the Max Planck in Heidelberg and has now been extensively tested on intact mouse and salamander retina and most recently on macaque retina. In collaboration with Peter Detwiler and his students we have successfully targeted both midget, parasol and other ganglion cells for whole cell recording in the intact macaque retina by imaging ganglion cell bodies and making whole-cell recordings after mechanical removal of the inner limiting membrane. Midget and parasol cells showed characteristic and long lasting light responses in this recording configuration. The cell bodies and dendritic trees were then intracellularly loaded with Calcium Green via the recording pipette and dendritic branches were targeted for imaging calcium signals in response to diffuse light pulses. As found previously for salamander and mouse ganglion cells, macaque ganglion cell dendrites show clear and rapid changes in calcium concentration in response to light steps. Finally, we have incorporated a new visual stimulus, using a digital light projector, which will permit the application of cone-specific visual stimuli during dendritic recording.