A new Opn4cre recombinase mouse line to target intrinsically photosensitive retinal ganglion cells (ipRGCs).

Intrinsically photosensitive retinal ganglion cells (ipRGCs) play a crucial role in several physiological light responses. In this study we generate a new Opn4cre knock-in allele (Opn4cre(DSO)), in which cre is placed immediately downstream of the Opn4 start codon. This approach aims to faithfully reproduce endogenous Opn4 expression and improve 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). Using this new tool, we describe the topographical distributions of ipRGC types across the retinal landscape, uncovering distinct ventronasal biases for M5 and M6 types, setting them apart from their M1-M4 counterparts. In the brain, we find vastly different labeling patterns between lines, with Opn4cre(DSO) only labeling ipRGC axonal projections to their targets. The combination of off-target effects of Opn4cre(Saha) across the retina and brain, coupled with diminished efficiencies of both Cre lines when coupled to less sensitive reporters, underscores the need for careful consideration in experimental design and validation with any Opn4cre driver. 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.

Light-dependent phosphorylation of the carboxy tail of mouse melanopsin.

Melanopsin-based phototransduction is involved in non-image forming light responses including circadian entrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep in vertebrates. Given that the functions of melanopsin involve the measurement and summation of total environmental luminance, there would appear to be no need for the rapid deactivation typical of other G-protein coupled receptors. In this study, however, we demonstrate that heterologously expressed mouse melanopsin is phosphorylated in a light-dependent manner, and that this phosphorylation is involved in regulating the rate of G-protein activation and the lifetime of melanopsin's active state. Furthermore, we provide evidence for light-dependent phosphorylation of melanopsin in the mouse retina using an in situ proximity ligation assay. Finally, we demonstrate that melanopsin preferentially interacts with the GRK2/3 family of G-protein coupled receptor kinases through co-immunoprecipitation assays. Based on the complement of G-protein receptor kinases present in the melanopsin-expressing retinal ganglion cells, GRK2 emerges as the best candidate for melanopsin's cognate GRK.