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Orexinergic projections to substantia innominata mediate arousal and analgesia. BACKGROUND: Understanding neural circuits involved in anaesthesia is crucial for improving its safety and efficacy. Lateral hypothalamic area orexinergic neurones (LHAOX), projecting broadly, are essential in regulating arousal and pain. However, the precise targets remain unclear. Here we investigated the orexin projections to the substantia innominata (SI). METHODS: Combining optogenetics, fibre photometry, electroencephalography, and electromyography allowed us to manipulate orexin activities while simultaneously recording local ligand release and global cortical activities during isoflurane anaesthesia. Brain slice electrophysiology revealed the synaptic connections in SI, while RNAscope was used to examine the distribution of orexin receptors and downstream neuronal types. RESULTS: Presynaptic vesicles were identified in the orexin-releasing axon terminals in SI, where 49.2% of cells expressed orexin receptor-2 (OX2R) and 6.8% expressed orexin receptor-1 (OX1R). Orexin release in SI was reversibly suppressed by isoflurane. Optogenetic activation of the LHAOX→SI circuit increased orexin release and promoted arousal from various anaesthesia stages, including during isoflurane 0.75 vol% (P<0.0001), prolongation of isoflurane 3 vol% induction (P=0.0033), and acceleration of emergence from isoflurane 2 vol% (P<0.0001). Furthermore, activating this circuit induced analgesia to both thermal (P<0.0001) and inflammatory (P<0.0001) pain. Patch-clamp recordings revealed that optogenetic activation of orexin terminals in SI elicited excitatory postsynaptic currents, which were blocked by an OX2R antagonist. The SI contains more GABAergic (28.2%) and glutamatergic (12.0%) neurones than cholinergic neurones (4.1%), all of which expressed OX2R. CONCLUSIONS: Hypothalamic area orexinergic neurones innervate substantia innominata neurones to regulate both arousal and pain predominantly through orexin receptor-2.