Molecular and spatial analysis of ganglion cells on retinal flatmounts identifies perivascular neurons resilient to glaucoma.

Recent transcriptomic studies have categorized mouse retinal ganglion cells (RGCs) into 45 types; however, little is known about their spatial distributions on the two-dimensional retinal surface and how their local microenvironments impact their functions. Here, we optimized a workflow combining imaging-based spatial transcriptomics (multiplexed-error robust fluorescent in situ hybridization [MERFISH]) and immunostaining on retinal flatmounts. We computationally registered the somata distributions of all RGCs and found that 34/45 molecularly defined types exhibited non-uniform distributions. We analyzed local neighborhoods for each cell and identified seven RGC types enriched in the perivascular niche, including direction-selective RGC (DSGC) and intrinsically photosensitive RGC (ipRGC) types. We further examined an experimental glaucoma model and found that surviving RGCs are enriched in the perivascular niche. Perivascular DSGCs and M1 ipRGCs were especially resilient, suggesting that proximity to vasculatures confers mTOR-independent, cell-extrinsic neuroprotection. Together, our work provides a comprehensive spatial atlas of RGC types and links their microenvironment to differential vulnerability in neurodegeneration.