Dataset 2: Brief illumination of an optoGPCR elicits prolonged behavioral inhibition in Drosophila

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 Gαi/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.光控G蛋白偶联受体（optoGPCRs）是一类通过细胞内信号级联反应发挥功能的视蛋白。作为光遗传工具，双稳态抑制型optoGPCRs颇具应用价值，因其可将神经元沉默时长与光照时长解耦：仅需短暂的光脉冲即可触发持续的信号转导。来自斯氏按蚊的视蛋白3（MosOPN3）是一种偶联Gαi/o的双稳态视蛋白，可在哺乳动物神经元与秀丽隐杆线虫中产生持续的抑制效应，但它在醋蝇——黑腹果蝇中的应用潜力尚未得到验证。本研究在成年果蝇中测试了三种optoGPCRs；其中MosOPN3表现最为优异，并在运动行为与记忆实验中，与已成熟的抑制工具盖氏隐杆藻阴离子通道视紫红质1（Guillardia theta anion channelrhodopsin 1，GtACR1）进行了对照基准测试。在绝大多数测试的神经元类别中，MosOPN3的抑制效力与GtACR1相当，且发育毒性显著更低。在整个发育周期中将果蝇饲养于添加全反式视黄醛的培养基中，可同时提升两种视蛋白的存活率与抑制效能。单次绿光脉冲（最短仅需5秒）即可抑制果蝇行为长达数分钟，且足以在1分钟的训练周期中完全阻断厌恶性嗅觉学习。对于果蝇而言，MosOPN3是一种低毒性的光遗传抑制工具，可通过短暂的光脉冲实现长达数分钟的神经元沉默——尤其适用于持续激活光会干扰实验的研究场景。