PROJECT SUMMARY Functional retinal ganglion cell (RGC) replacement could restore vision to tens of millions of people who are blind from glaucoma and other optic neuropathies. Establishment of techniques for RGC differentiation from pluripotent stem cells and the achievement of long-distance endogenous axon regeneration within the optic nerve support the promise of RGC replacement therapies. However, clinical translation requires significant improvements in the functional integration of transplanted RGCs into the host retinal neurocircuitry. To enable the study of synaptogenesis by transplanted neurons, we propose developing and validating an innovative, versatile, sensitive, experimental tool that leverages transsynaptic transport of wheat germ agglutinin (WGA) protein fused to Cre recombinase to enable expression of a Cre-dependent reporter in synaptically integrated donor neurons. The label is durable, facilitating downstream applications such as single cell isolation and transcriptomic analysis, and bidirectional to allow the labeling of either pre- or post-synaptic graft-host neuronal partners. Here, we propose to establish human pluripotent cell lines to be used in conjunction with highly efficient recombinant AAV vectors to report the functional retinal of transplanted RGCs. Since expression of the fluorescent reporter is automatic upon genomic recombination, the tool will facilitate real-time detection of functional neuronal integration in vivo. We propose a short wavelength (blue) fluorescent reporter for synaptogenesis, which will be compatible with additional red and green fluorescence for multicolor microscopy and ophthalmoscopy. We will characterize the kinetics of expression and validate the specificity of this synaptogenesis reporter tool using a combination of high-resolution confocal microscopy, immunolabeling of synaptic machinery, and single-cell electrophysiology within retinal flatmounts. Further, we will study the functional integration of transplanted human RGCs in vivo using a custom-built multicolor adaptive optics scanning laser ophthalmoscope. The instrumentation provides subcellular resolution within in the xy plane and depth discrimination with the retina through z-stacking. Longitudinal imaging of living eyes post- transplantation will therefore enable the correlation of donor RGC dendrites targeting of the inner plexiform layer with reporting of functional synaptogenesis. We will characterize the structural events leading to retinal integration of donor RGCs. This work will provide a foundation for future experimentation that includes manipulation of the host microenvironment to improve engraftment efficiency, in vivo optical electrophysiology of integrated RGCs, and robust analyses of RGC interactions with various resident retinal cells using cell-specific reporter mice.