Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat.

Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3(85-101)). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [125I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.

Comparative localization of leucine-rich repeat-containing G-protein-coupled receptor-7 (RXFP1) mRNA and [33P]-relaxin binding sites in rat brain: restricted somatic co-expression a clue to relaxin action?

Relaxin is a polypeptide hormone with established actions associated with reproductive physiology, but until recently the precise nature of the relaxin receptor and its transmembrane signaling mechanisms had remained elusive. In 2002 however, the leucine-rich-repeat-containing G-protein-coupled receptor-7 (now classified as RXFP1) was identified as a cognate receptor for relaxin, with activation resulting in stimulation of intracellular cAMP production. These findings, along with the presence and putative actions of relaxin within the CNS and earlier descriptions of relaxin binding sites in brain, suggest the importance and feasibility of determining if these relaxin binding sites represent leucine-rich-repeat-containing G-protein-coupled receptor-7 and their precise comparative distribution. Thus, the current study reports the distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the rat brain using in situ hybridization histochemistry of [(35)S]-labeled oligonucleotides and the comparative distribution of [(33)P]-human relaxin binding sites. The extensive, topographical distribution of leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA throughout the adult rat brain correlated very closely to that of [(33)P]-relaxin binding sites. Leucine-rich-repeat-containing G-protein-coupled receptor-7 mRNA was expressed by neurons in several brain regions, including the olfactory bulb, cerebral cortex, thalamus, hippocampus, hypothalamus, midbrain, pons and medulla. Receptor transcripts were most abundant in areas such as the basolateral amygdala, subiculum, deep layers of the cingulate, somatosensory and motor cortices and intralaminar/midline thalamic nuclei. These areas also contained very high densities of [(33)P]-relaxin binding sites, suggesting a largely somatic localization of leucine-rich-repeat-containing G-protein-coupled receptor-7 protein and site of action for relaxin peptide. The central distribution of relaxin-producing neurons has been described, while data on the topography of nerve terminals that contain and secrete the peptide are currently lacking; but overall these findings strongly suggest that leucine-rich-repeat-containing G-protein-coupled receptor-7 is the cognate receptor for relaxin in the rat brain, and support a role for relaxin-leucine-rich-repeat-containing G-protein-coupled receptor-7 signaling in various somatosensory, autonomic and neurohumoral pathways, which warrants further investigation.

Circulating relaxin acts on subfornical organ neurons to stimulate water drinking in the rat.

Relaxin, a peptide hormone secreted by the corpus luteum during pregnancy, exerts actions on reproductive tissues such as the pubic symphysis, uterus, and cervix. It may also influence body fluid balance by actions on the brain to stimulate thirst and vasopressin secretion. We mapped the sites in the brain that are activated by i.v. infusion of a dipsogenic dose of relaxin (25 microg/h) by immunohistochemically detecting Fos expression. Relaxin administration resulted in increased Fos expression in the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), median preoptic nucleus, and magnocellular neurons in the supraoptic and paraventricular nuclei. Ablation of the SFO abolished relaxin-induced water drinking, but did not prevent increased Fos expression in the OVLT, supraoptic or paraventricular nuclei. Although ablation of the OVLT did not inhibit relaxin-induced drinking, it did cause a large reduction in Fos expression in the supraoptic nucleus and posterior magnocellular subdivision of the paraventricular nucleus. In vitro single-unit recording of electrical activity of neurons in isolated slices of the SFO showed that relaxin (10(-7) M) added to the perfusion medium caused marked and prolonged increase in neuronal activity. Most of these neurons also responded to 10(-7) M angiotensin II. The data indicate that blood-borne relaxin can directly stimulate neurons in the SFO to initiate water drinking. It is likely that circulating relaxin also stimulates neurons in the OVLT that influence vasopressin secretion. These two circumventricular organs that lack a blood-brain barrier may have regulatory influences on fluid balance during pregnancy in rats.