OXYTOCIN SIGNALLING IN THE NUCLEUS INCERTUS: IMPLICATIONS FOR TRANSVENTRICULAR AROUSAL MODULATION

Oxytocin (OT) and its receptor (OTR) regulate diverse physiological and behavioural processes, including social interaction, reproduction, homeostasis, learning, and emotional regulation. Although OT is well established as a modulator of social behaviour, its role in brain arousal, an essential state for attention and adaptive behaviour, remains poorly understood. The nucleus incertus (NI), a brainstem structure implicated in arousal control, also regulates several functions influenced by OT/OTR signalling. Here, we investigated whether OT/OTR signalling modulates NI activity and contributes to arousal regulation. To characterize OT/OTR signalling within the rat NI we included immunohistochemistry, fluorescent in situ hybridisation, viral anatomical tracing, ex vivo electrophysiology, chemogenetic manipulation during behavioural tasks, fibre photometry, and pupillometry. We found that 88% of OTR-expressing NI neurons are GABAergic, and that all relaxin-3 and cholecystokinin neurons in the NI co-express OTR mRNA. Electrophysiological recordings revealed a dose-dependent OTR-mediated excitatory effect of OT in approximately 70% of NI neurons. Chemogenetic activation of NI OTR neurons increased locomotor activity, indicating a role in behavioural arousal. Viral tracing and immunohistochemistry revealed no direct oxytocinergic innervation of the NI, supporting cerebrospinal fluid–mediated volume transmission. Consistently, intracerebroventricular OT administration induced pupil dilation, and OT sensor imaging confirmed OT diffusion to the NI. Together, these findings indicate that NI OTR neurons participate in arousal regulation and support the hypothesis of CSF-mediated OT signalling as a neuromodulatory pathway within this brainstem region. These insights extend our understanding of neuropeptide signalling in the brain and its influence on behaviour.