Brief illumination of an optoGPCR elicits prolonged inhibition of Drosophila behavior

Light-actuated G protein-coupled receptors (optoGPCRs) are a class of opsins that act through intracellular signaling cascades. As optogenetic tools, bistable inhibitory optoGPCRs are attractive as they decouple the duration of silencing from the duration of illumination: brief light pulses drive prolonged signaling. Opsin3 from the mosquito Anopheles stephensi (MosOPN3) is a bistable Gi/o-coupled opsin that produces sustained inhibition in mammalian neurons and C. elegans, but its utility in vinegar flies, Drosophila melanogaster, remains untested. Here we tested three optoGPCRs in adult flies; MosOPN3 emerged as the most effective and was benchmarked against an established inhibitor, Guillardia theta anion channelrhodopsin 1 (GtACR1), in walking and memory assays. MosOPN3 inhibitory potency was comparable to that of GtACR1 across most neuronal classes tested, with substantially lower developmental toxicity. Rearing flies on all-trans-retinal-supplemented food throughout development improved both viability and inhibitory efficacy for both opsins. A single pulse of green light (as brief as 5 s) inhibited behavior for minutes, and was sufficient to ablate aversive olfactory learning over a one-minute training epoch. For flies, MosOPN3 is a low-toxicity optogenetic inhibitor for flies that yields minutes of silencing from brief light pulses--especially useful where continuous actuating light would be confounding. Key pointsO_LITo our knowledge, Anopheles stephensi Opsin3 (OPN3) is the first GPCR opsin that inhibits behavior in adult Drosophila melanogaster. C_LIO_LIOPN3 has inhibitory efficacy close to that of GtACR1, with superior viability. C_LIO_LIBrief light pulses drive sustained inhibition lasting minutes, eliminating the need for continuous illumination. C_LIO_LIFor both GtACR1 and OPN3, rearing flies on ATR-supplemented food enhances inhibition efficacy and improves developmental viability. C_LI