Relaxin receptor activation in the basolateral amygdala impairs memory consolidation.

The peptide-hormone relaxin has well-established actions in male and female reproductive tracts, and has functional effects in circumventricular regions of brain involved in neurohormonal secretion. In the current study, we initially mapped the distribution of mRNA encoding the relaxin receptor--leucine-rich repeat-containing G-protein-coupled receptor 7 (LGR7)- and [33P]-human relaxin-binding sites in extra-hypothalamic sites of male Sprague-Dawley rats. The basolateral amygdala (BLA) expressed high levels of LGR7 mRNA and relaxin-binding sites and, although relaxin peptide was not detected in the BLA, several brain regions that send projections to the BLA were found to contain relaxin-expressing neurons. As it is well established that the BLA is involved in regulating the consolidation of memory for emotionally arousing experiences, we investigated whether activation of LGR7 in the BLA modulated memory consolidation for aversively motivated inhibitory avoidance training. Bilateral infusions of human relaxin (10-200 ng in 0.2 microL) into the BLA immediately after inhibitory avoidance training impaired 48-h retention performance in a dose-dependent manner. Delayed infusions of relaxin into the BLA 3 h after training were ineffective, indicating that the retention impairment was due to influences on memory consolidation. Post-training infusions of relaxin into the adjacent central amygdala, which is devoid of LGR7, did not impair retention. These findings suggest a novel function for endogenous relaxin-LGR7 signalling in rat brain involving regulation of memory consolidation.

Localization of LGR7 gene expression in adult mouse brain using LGR7 knock-out/LacZ knock-in mice: correlation with LGR7 mRNA distribution.

Knowledge of the distribution of the relaxin receptor, LGR7, in the brain provides a basis for studies of the physiologic actions of relaxin. LGR7 knock-out (KO) mice were produced by the in-frame replacement of LGR7 exon 10 and 11 with a LacZ-reporter cassette (knock-in [KI]), and in this study we used LGR7-KO/LacZ-KI mice to determine the regional/cellular distribution of LGR7 gene expression in adult mouse brain by assessing beta-galactosidase activity in perfusion-fixed sections. High densities of beta-galactosidase-positive neurons were detected in anterior olfactory and claustrum/endopiriform nuclei, deep layers of cortex (particularly somatosensory), and the subiculum. Low to moderate densities were detected in olfactory bulb (periglomerular layer), cingulate cortex, subfornical organ, hippocampal CA2/dentate hilus, amygdala, hypothalamus, and thalamus. This LGR7/LacZ expression appears to recapitulate that of native LGR7 in wild-type mice and provides a model to further investigate the phenotype of LGR7-responsive neurons in the brain and to help reveal functions associated with central relaxin signaling.

Localization of LGR7 (relaxin receptor) mRNA and protein in rat forebrain: correlation with relaxin binding site distribution.

Discrete neuronal populations in brain express relaxin and relaxin-3, and molecular studies have identified former-orphan, G-protein-coupled receptors LGR7 and GPCR135 as their native receptors. To better understand the role of central relaxin systems, we began to assess the anatomic distribution of these receptors and ligands in brain. This study documents the widespread distribution of LGR7 mRNA and LGR7-like immunoreactivity (LI) throughout adult rat forebrain areas shown to contain specific [33P]-relaxin binding sites. High densities of LGR7 mRNA hybridization were detected in deep layers of neocortex, hypothalamic paraventricular and supraoptic nuclei and within hippocampal subiculum and CA3, the basolateral amygdala and subfornical organ. Low to moderate hybridization was detected in septum, midline thalamic nuclei, arcuate and supramammillary nuclei, and regions of the midbrain pons. Complementary expression of LGR7-LI was observed in cortical pyramidal neurons, hypothalamic magnocellular neurons, and hippocampal pyramidal and interneurons. These findings provide further evidence for actions of relaxin as a modulator in somatosensory, autonomic, and neuroendocrine pathways.

Relaxin: new peptides, receptors and novel actions.

Relaxin has long been known as a hormone of pregnancy. Until recently, little was known of potential roles for relaxin in non-pregnant females and males. The identification of a new gene encoding relaxin-3 (RLN3), the discovery of the elusive relaxin receptor and a novel role for relaxin-1 in regulating the normal turnover of collagen has provided us with unique insights into potential new roles for this peptide family. The Rln3 gene appears to be predominantly expressed in the brain, and mapping studies indicate a highly developed network of Rln3, Rln1 and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides might have important roles in the central nervous system. Rln1-knockout mice show progressive tissue fibrosis as they age, and this fibrosis leads to functional changes in both the heart and lungs. Hence, the biological significance of this enigmatic peptide family is expanding, as are its potential clinical uses.

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain.

Relaxin is a peptide hormone with known actions associated with female reproductive physiology, but it has also been identified in the brain. Only one relaxin gene had been characterized in rodents until recently when a novel human relaxin gene, human gene-3 (H3) and its mouse equivalent (M3) were identified. The current study reports the identification of a rat homologue, rat gene-3 (R3) relaxin that is highly expressed in a discrete region of the adult brain. The full R3 relaxin cDNA was generated using RT-PCR and 3' and 5' RACE protocols. The derived amino acid sequence of R3 relaxin retains all the characteristic features of a relaxin peptide and has a high degree of homology with H3 and M3 relaxin. The distribution of R3 relaxin mRNA in adult rat brain was determined and highly abundant expression was only detected in neurons of the ventromedial dorsal tegmental nucleus (vmDTg) in the pons, whereas all other brain areas were unlabelled or contained much lower mRNA levels. Relaxin binding sites and relaxin immunoreactivity were also detected in the vmDTg. These together with earlier findings provide strong evidence for a role(s) for multiple relaxin peptides as neurotransmitters and/or modulators in the rat CNS.

Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family.

We have identified a novel human relaxin gene, designated H3 relaxin, and an equivalent relaxin gene in the mouse from the Celera Genomics data base. Both genes encode a putative prohormone sequence incorporating the classic two-chain, three cysteine-bonded structure of the relaxin/insulin family and, importantly, contain the RXXXRXX(I/V) motif in the B-chain that is essential for relaxin receptor binding. A peptide derived from the likely proteolytic processing of the H3 relaxin prohormone sequence was synthesized and found to possess relaxin activity in bioassays utilizing the human monocytic cell line, THP-1, that expresses the relaxin receptor. The expression of this novel relaxin gene was studied in mouse tissues using RT-PCR, where transcripts were identified with a pattern of expression distinct from that of the previously characterized mouse relaxin. The highest levels of expression were found in the brain, whereas significant expression was also observed in the spleen, thymus, lung, and ovary. Northern blotting demonstrated an approximately 1.2-kb transcript present in mouse brain poly(A) RNA but not in other tissues. These data, together with the localization of transcripts in the pars ventromedialis of the dorsal tegmental nucleus of C57BLK6J mouse brain by in situ hybridization histochemistry, suggest a new role for relaxin in neuropeptide signaling processes. Together, these studies describe a third member of the human relaxin family and its equivalent in the mouse.