FROM RETINA TO LGN: CELL-SPECIFIC MULTICOLOR MAPPING OF OPTOGENETIC VISION RESTORATION PATHWAYS IN NON-HUMAN PRIMATES

Background: As optogenetic strategies for vision restoration advance toward clinical application, including human trials, it is essential to define the retinal cell types targeted by gene delivery and to determine how restored signals are integrated into central visual pathways. Characterizing the diversity, spatial distribution, and connectivity of targeted retinal ganglion cells (RGCs) is critical for evaluating the functional potential of these approaches.

Methods: We examined the RGC population transduced by optogenetic gene delivery in the foveal and parafoveal retina of non-human primates. Using the same AAV construct previously employed in human and primate studies, we implemented a Brainbow-like multicolor labeling strategy to achieve stochastic, cell-specific fluorescent tagging of RGCs. This approach enabled high-resolution three-dimensional reconstruction of individual neurons within densely labeled tissue and allowed tracing of their axonal projections to central targets.

Results: Multicolor labeling revealed diversity among targeted RGCs, identifiable by unique fluorophore combinations, and enabled precise quantification of their density and spatial organization in the parafoveal retina. Labeled RGC axons were traced to the lateral geniculate nucleus (LGN), where VGlut2 immunostaining identified glutamatergic synaptic terminals within retinorecipient layers. Three-dimensional reconstructions allowed measurement of the LGN volume receiving direct retinal input and analysis of input density across magnocellular, parvocellular, and koniocellular subdivisions, each exhibiting distinct spatial organization patterns.

Interpretation: This multicolor mapping strategy provides a framework for dissecting retinal and post-retinal circuitry in primates. By linking cell-level retinal targeting to layer-specific central connectivity, it offers mechanistic insight into how optogenetically restored visual signals may be processed beyond the retina.