Defining spatial nonuniformities of all ipRGC types using an improved Opn4^cre recombinase mouse line

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study, we generate an improved Opn4^cre knockin allele (Opn4^cre(DSO)), which faithfully reproduces endogenous Opn4 expression and improves compatibility with widely used reporters. We evaluated the efficacy and sensitivity of Opn4^cre(DSO) for labeling in retina and brain and provide an in-depth comparison with the extensively utilized Opn4^cre(Saha) line. Through this characterization, Opn4^cre(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 Opn4^cre(DSO) line labels ipRGC projections with minimal labeling of cell bodies. Overall, the Opn4^cre(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.