Relaxin-3 (RLX3), a newly identified member of the relaxin peptide family, is distinguished by its enriched expression in GABA projection neurons of the pontine nucleus incertus (NI), which are postulated to participate in forebrain neural circuits involved in behavioural activation and stress responses. In this regard, corticotrophin-releasing factor-1 receptor (CRF(1)) is abundantly expressed by NI neurons; central CRF administration activates c-fos expression in NI; and various stressors have been reported to increase NI neuron activity. In studies to determine whether a specific neurogenic stressor would activate RLX3 expression, we assessed the effect of a repeated forced swim (RFS) on levels of RLX3 mRNA and heteronuclear (hn) RNA in rat NI by in situ hybridization histochemistry of exon- and intron-directed oligonucleotide probes, respectively. Exposure of rats to an RFS (10 min at 23 degrees C, 24 h apart), markedly increased RLX3 mRNA levels in NI at 30-60 min after the second swim, before a gradual return to basal levels over 2-4 h, while RLX3 hnRNA levels were significantly up-regulated at 60-120 min post-RFS, following a transient decrease at 30 min. Systemic treatment of rats with a CRF(1) antagonist, antalarmin (20 mg/kg, i.p.) 30 min prior to the second swim, blunted the stress-induced effects on RLX3 transcripts. Relative levels of RLX3-immunostaining in NI neurons appeared elevated at 3 h post-swim, but not at earlier time points (30-60 min). These results suggest that acute stress-induced CRF secretion can rapidly alter RLX3 gene transcription by activation of CRF(1) present on NI neurons. More generally, these studies support a role for RLX3 neural networks in the normal neural and physiological response to neurogenic stressors in the rat.