The ventral hippocampus (vHPC) is a brain locus centrally involved in the control of stress and anxiety, with dysfunction of its interneuron network strongly implicated in clinical anxiety-related disorders. The vHPC is heavily innervated by neurons in the pontine tegmental, nucleus incertus (NI) – the primary source of the neuropeptide, relaxin-3 (RLN3), in the rat brain. Intriguingly, sustained activation of the RLN3 receptor, RXFP3, in the vHPC of rats increased anxiety and social avoidance [1], yet the precise underlying neuronal mechanisms remain unknown. Therefore, this study investigated the neurochemical profile and functional connectivity of the NI-vHPC pathway in rat brain, and conducted comparative anatomical studies in human hippocampus. Immunohistochemical staining of the rat hippocampus revealed an abundant innervation of the vHPC dentate gyrus (vDG) by RLN3-immunoreactive nerve fibres. In addition, viral-based neural tract-tracing identified a predominantly ipsilateral innervation of the rat vDG by RLN3 NI neurons. HiPlex in situ hybridization (ISH) studies revealed that RXFP3 mRNA-expressing neurons in the rat vDG, co-expressed vesicular GABA transporter (vGAT) and somatostatin mRNA. Notably, ISH with human anterior hippocampal sections also revealed co-localization of RXFP3 and somatostatin mRNA. Finally, ex vivo multi-electrode array recordings demonstrated an inhibitory effect of RXFP3 activation on the rat vDG network activity. These findings indicate a direct influence of RLN3 on GABAergic vDG interneurons in rat and human, which may underlie the effects of RLN3/RXFP3 signalling on anxiety-related behaviours.1.Rytova V et al. (2019) Hippocampus 29, 905-920.Funding: National_Science_Centre_Poland UMO-2023/49/B/NZ4/01885, UMO-2018/30/E/NZ4/00687; MiniGrant2023 ID.UJ; BrightFocus Foundation A2021006; Alzheimer’s Association AARFD-22-972099.