Early-life stress (ELS) disrupts brain development and predisposes sufferers to stress-related disorders and compulsive behaviour in adulthood. Accumulating evidence connects these disorders with neurons within the stress-sensitive, brainstem nucleus incertus (NI), which synthesize the neuropeptides, relaxin-3 (RLN3) and cholecystokinin (CCK). Although NI RLN3 neurons are implicated in stress responses, arousal and drug-seeking behaviour, their vulnerability to stress during development has not been explored, and little is known about NI CCK neurons. Therefore, this study investigated the influence of maternal separation (MS), an established model of ELS, on NI neuronal populations.Adult, male Sprague-Dawley rats from control and MS groups were subjected to acute restraint stress and subsequent immunohistochemical staining of NI neurons. Separate control and MS rats were used for in situ hybridisation or whole-cell, patch-clamp recordings, combined with dendritic tracing and morphological assessment of NI neurons.MS induced multi-level and cell-type specific changes in NI neurons, including altered activation in response to acute stress, modified active and passive membrane properties, and altered action potential properties. Moreover, MS produced a remodelling of the dendritic tree of NI CCK neurons, and altered the expression of stress-related, CRHR1 and TrkA receptor mRNA in the NI.MS-induced molecular, structural and functional changes within NI neurons may contribute to the development of compulsive behaviours and other ELS-related disorders; and the role of MS stress-sensitive NI CCK neurons, in particular, needs to be further explored.Funding: National Science Centre, Poland, UMO-2018/30/E/NZ4/00687, UMO-2023/49/B/NZ4/01885, UMO-2017/27/N/NZ4/01545.