Relaxin-3/RXFP3 signalling in mouse hypothalamus: no effect of RXFP3 activation on corticosterone, despite reduced presynaptic excitatory input onto paraventricular CRH neurons in vitro.

Relaxin-3/RXFP3 signalling is proposed to be involved in the neuromodulatory control of arousal- and stress-related neural circuits. Furthermore, previous studies in rats have led to the proposal that relaxin-3/RXFP3 signalling is associated with activation of the hypothalamic-pituitary-adrenal axis, but direct evidence for RXFP3-related actions on the activity of hypothalamic corticotropin-releasing hormone (CRH) neurons is lacking. In this study, we investigated characteristics of the relaxin-3/RXFP3 system in mouse hypothalamus. Administration of an RXFP3 agonist (RXFP3-A2) intra-cerebroventricularly or directly into the paraventricular nucleus of hypothalamus (PVN) of C57BL/6J mice did not alter corticosterone levels. Similarly, there were no differences between serum corticosterone levels in Rxfp3 knockout (C57BL/6JRXFP3TM1) and wild-type mice at baseline and after stress, despite detection of the predicted stress-induced increases in serum corticosterone. We examined the nature of the relaxin-3 innervation of PVN in wild-type mice and in Crh-IRES-Cre;Ai14 mice that co-express the tdTomato fluorophore in CRH neurons, identifying abundant relaxin-3 fibres in the peri-PVN region, but only sparse fibres associated with densely packed CRH neurons. In whole-cell voltage-clamp recordings of tdTomato-positive CRH neurons in these mice, we observed a reduction in sEPSC frequency following local application of RXFP3-A2, consistent with an activation of RXFP3 on presynaptic glutamatergic afferents in the PVN region. These studies clarify the relationship between relaxin-3/RXFP3 inputs and CRH neurons in mouse PVN, with implications for the interpretation of current and previous in vivo studies and future investigations of this stress-related signalling network in normal and transgenic mice, under normal and pathological conditions.