Defining spatial nonuniformities of all ipRGC types using an improved Opn4crerecombinase mouse line

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study, we generate an improved Opn4creknockin allele (Opn4cre(DSO)), which faithfully reproduces endogenous Opn4expression and improves compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4cre(DSO)for labeling in retina and brain and provide an in-depth comparison with the extensively utilized Opn4cre(Saha)line. Through this characterization, Opn4cre(DSO)demonstrated higher specificity in labeling ipRGCs with minimal recombination escape. Leveraging a combination of electrophysiological, molecular, and morphological analyses, we confirmed its sensitivity in detecting all ipRGC types (M1–M6) and defined their unique topographical distribution across the retina. In the brain, the Opn4cre(DSO)line labels ipRGC projections with minimal labeling of cell bodies. Overall, the Opn4cre(DSO)mouse line represents an improved tool for studying ipRGC function and distribution, offering a means to selectively target these cells to study light-regulated behaviors and physiology.