RELAXIN-3 IN GABA PROJECTION NEURONS OF NUCLEUS INCERTUS SUGGESTS WIDESPREAD INFLUENCE ON FOREBRAIN CIRCUITS VIA G-PROTEIN-COUPLED RECEPTOR-135 IN THE RAT S. MA, a * P. BONAVENTURE, d T. FERRARO, a P.-J. SHEN, a T. C. D. BURAZIN, a R. A. D. BATHGATE, a,b C. LIU, d G. W. TREGEAR, a,b S. W. SUTTON d AND A. L. GUNDLACH a,c a Howard Florey Institute, The University of Melbourne, Melbourne, Victoria 3010, Australia b Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Victoria, Australia c Department of Anatomy and Cell Biology, The University of Mel- bourne, Melbourne, Victoria, Australia d Neuroscience Group, Johnson & Johnson Pharmaceutical Research and Development, LLC, San Diego, CA, USA Abstract—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 phar- macological 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 recep- tor; RXFP3). In ongoing studies to understand the physiolog- ical functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immuno- histochemistry, using an affinity-purified polyclonal anti- serum 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 cortico- tropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggest- ing a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus in- certus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, sep- tum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lat- eral hypothalamus/medial forebrain bundle and ventral hip- pocampus; 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 [ 125 I]-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 neuroendo- crine control of metabolism and reproduction and higher- order processes such as stress and cognition. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: relaxin-3 like-immunohistochemistry, GPCR135 mRNA, nucleus incertus efferents, stress, septo-hippocam- pal theta rhythm. The insulin/relaxin peptide superfamily consists of a large number of peptides expressed in a wide range of species (Wilkinson et al., 2005; Bathgate et al., 2006a). Relaxin-3 (RLX3), first identified in 2002 by our laboratory (Bathgate et al., 2002; Burazin et al., 2002), is the latest addition to this family; and in contrast to relaxin that is expressed in several tissues, especially the female reproductive tract, RLX3 is primarily expressed in the brain of human, mouse and rat (Bathgate et al., 2002, 2003; Burazin et al., 2002). In the rodent brain, RLX3 mRNA is enriched in a midline pontine region embedded in the caudal ventromedial cen- tral gray known as the nucleus incertus (NI) (Bathgate et al., 2002; Burazin et al., 2002), which is a distinct cell group that consists of two parts, the midline compacta (NIc) and the dissipata (NId) that extends laterally from the NIc (Goto et al., 2001; Olucha-Bordonau et al., 2003). Renewed interest in the anatomy and physiology of the NI was initially aroused by its high level of expression of cortico- tropin-releasing factor receptor-1 (CRF-R1) mRNA, and a belief that it might mediate some of the extra-pituitary actions of CRF in response to stress (Potter et al., 1994; Bittencourt and Sawchenko, 2000). Resultant anterograde and retrograde tract-tracing studies of the NI suggested that this nucleus is well positioned to integrate information relating to memory and attentional state (Goto et al., 2001; Olucha-Bordonau et al., 2003) and concurrent in vivo stud- ies in rats reported that stressors such as forced swimming or restricted food access increased Fos expression in NI neurons (Goto et al., 2001, 2005). Extensive genetic, molecular and pharmacological studies have now shown that the preferred native receptor for RLX3 is G-protein-coupled receptor-135 (GPCR135 or somatostatin- and angiotensin-like peptide receptor (SALPR); now classified as relaxin family peptide-3 recep- *Corresponding author. Tel: +61-3-8344-6759; fax: +61-3-9347-0446. E-mail address: s.ma@hfi.unimelb.edu.au (S. Ma). Abbreviations: CRF, corticotropin-releasing factor; CRF-R1, cortico- tropin-releasing factor receptor-1; DAB, 3,3=-diaminobenzidine; DR, dorsal raphe nucleus; GAD65, glutamic acid decarboxylase-65; GPCR135, G-protein-coupled receptor-135; GPCR142, G-protein- coupled receptor-142; IGL, intergeniculate leaflet; LH, lateral hypothal- amus; -LI, -like immunoreactivity; LPO, lateral preoptic area; LS, lat- eral septum; MPO, medial preoptic area; MS, medial septal nucleus; NDB, nucleus of the diagonal band; NHS, normal horse serum; NI, nucleus incertus; NIc, nucleus incertus compacta; NId, nucleus incer- tus dissipata; PP, peripeduncular nucleus; PVN, paraventricular nu- cleus; RLX3, relaxin-3; VGlut2, vesicular glutamate transporter-2. Neuroscience 144 (2007) 165–190 0306-4522/07$30.00+0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.08.072 165