ANALYSIS OF OPN5 SIGNALING IN MOUSE AND HUMAN

Opsin 5 (OPN5; neuropsin) is a conserved violet/ultraviolet (UV)-sensitive G protein–coupled receptor (GPCR) in vertebrates. In mammals, OPN5 acts as a non-visual photopigment and is expressed in multiple extra-retinal tissues, including skin, testes, and several deep brain regions. However, its physiological roles and downstream signaling remain poorly understood. Here, we investigated OPN5 signaling using whole-cell patch-clamp recording and calcium imaging.
We first expressed C-terminal GFP-tagged mouse OPN5 in HEK293 cells in the presence of all-trans retinal (ATR). During voltage-clamp recordings, 60-s violet-light stimulation evoked slow, sustained currents. In calcium imaging, violet light decreased intracellular Ca²⁺ in cells expressing mouse OPN5 with GCaMP6s. In contrast, cells expressing human OPN5 with GCaMP8s showed an increase in intracellular Ca²⁺, indicating opposite calcium responses between mouse and human OPN5.
To examine OPN5 signaling in the brain, we generated OPN5-mGLx3 mice, in which the bright fluorescent protein mGreenLantern is knocked into the endogenous Opn5 locus to label OPN5-expressing cells. RNAscope in situ hybridization combined with immunohistochemistry confirmed that mGreenLantern expression was restricted to Opn5 mRNA–positive cells in the preoptic area (POA). In acute POA slices, mGreenLantern-positive neurons responded to violet-light stimulation in both the presence and absence of tetrodotoxin (TTX). Notably, violet light–evoked responses were abolished in homozygous mGLx3 mice, in which Opn5 is genetically deleted.
Together, these results reveal species-dependent OPN5 signaling and demonstrate that POA OPN5-expressing neurons directly respond to violet light via OPN5.