Relaxin-3, a peptide abundantly expressed by GABAergic neurons in the nucleus incertus, preferentially activates a Gi-protein-coupled receptor, RXFP3. Relaxin-3 neurons are stress-responsive, and constitute a conserved ascending network in rodent and primate brain, enriched in limbic areas involved in stress, arousal and emotion-related behaviours, such as hypothalamus, extended amygdala, ventral hippocampus (vHPC) and prefrontal cortex. Our research in rats has shown relaxin-3/RXFP3 signalling modulates arousal, sucrose/alcohol seeking [1], and depressive-/anxiety-like behaviours [2]. Our current goal is to characterize how relaxin-3/RXFP3 signalling in various limbic regions modulates ‘affective’ behaviour including innate anxiety and learned fear in the rat. We first examined effects of a specific RXFP3 agonist, RXFP3-A2, in an auditory fear conditioning paradigm. Central RXFP3 activation by intracerebroventricular (icv) injection of RXFP3-A2 (5-15 µg) in adult Sprague-Dawley rats (n=5-7/group) resulted in dose-related impairment of fear extinction associated with altered neural activation (Fos-staining) patterns in regions including PVN and amygdala. In contrast, icv RXFP3-A2 injection reduced innate anxiety in the elevated plus maze (EPM) and light/dark box (LDB) tests, and depressive-like indices (immobility) in a forced swim test [2], suggesting complex ‘topographic’ effects of RXFP3 signalling on fear, mood and anxiety, related to precise site(s) and timing of endogenous and exogenous peptide action. Therefore, we injected AAV1/2 constructs, which drive local secretion of RXFP3 agonist (R3/I5) or antagonist (R3(B1-22)R), into vHPC (n=6/group), and found that chronic RXFP3 activation decreases time spent (~90% decrease, p=0.011) and distance travelled (~90% decrease, p=0.008) in the light zone of the LDB c.f. control vector. We are currently assessing the impact of chronic modulation of vHPC RXFP3 on social interaction, spatial learning, and fear conditioning. These studies will better characterize the impact of this novel neuropeptide/receptor system and associated neural circuits on complex behaviours, with implications for understanding anxiety/mood disorders and potential therapeutic targets.