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?

Neuroscience 141 (2006) 329 –344

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? S. MA,a,b* P.-J. SHEN,a T. C. D. BURAZIN,a G. W. TREGEARa,b AND A. L. GUNDLACHa,c

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 Gprotein-coupled receptor-7 signaling in various somatosensory, autonomic and neurohumoral pathways, which warrants further investigation. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

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 Melbourne, Melbourne, Victoria, Australia

Key words: insulin/relaxin peptide family, G-protein-coupled receptor, olfaction, cognition, in situ hybridization histochemistry, radioligand autoradiography.

Abstract—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 Gprotein-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 [35S]-labeled oligonucleotides and the comparative distribution of [33P]-human relaxin binding sites. The extensive, topographical distribution of leucine-rich-repeat-containing G-proteincoupled receptor-7 mRNA throughout the adult rat brain correlated very closely to that of [33P]-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 [33P]-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

Relaxin is a 6-kDa polypeptide hormone and a member of the insulin superfamily with well-characterized endogenous synthesis and biological effects in reproductive tissues, particularly during parturition, in rodents, primates and man. In contrast, the effects of relaxin in non-reproductive tissues such as brain have for many years been less clear, largely due to a limited number of investigations. Such studies were until recently further restrained by a lack of knowledge about the precise nature of the relaxin receptor and its transmembrane signaling mechanisms. In 2002 however, two ‘orphan receptors’ were identified as likely relaxin receptors, namely the leucine-rich repeatcontaining G-protein-coupled receptor-7 and -8 (LGR7/8; Hsu et al., 2002). LGR7 and LGR8 were initially classified as members of the receptor superfamily for glycoprotein hormones including luteinizing hormone, follicle-stimulating hormone and thyrotropin-stimulating hormone (Hsu et al., 2000), but were subsequently discovered, when transfected into human embryonic kidney cells, to bind human relaxin with high affinity to produce stimulation of cAMP accumulation (Hsu et al., 2002). These findings and the simultaneous discovery of a further member of the insulin/relaxin peptide family—relaxin-3 (Bathgate et al., 2002; Burazin et al., 2002)—together with the subsequent discovery of additional candidate receptors and their likely native pairing with different insulin-like peptide ligands (see below) have rapidly increased our knowledge of the molecular pharmacology of multiple relaxin peptides and receptors (see Bathgate et al., 2006a) and laid the foundation for the better design and interpretation of further anatomical and functional studies of relaxins in the CNS. Thus, three genes have been identified that encode human relaxin peptides, namely RLN1 (Hudson et al., 1983), RLN2 (Crawford et al., 1984), and RLN3 (Bathgate et al., 2002); while the RLN2 gene homologue in mouse and rat is designated Rln1 (Sherwood, 1979; Evans

*Correspondence to: S. Ma, Howard Florey Institute, The University of Melbourne, Melbourne, Victoria 3010, Australia. Tel: ⫹61-3-83446759; fax: ⫹61-3-9347-0446. E-mail address: [email protected] (S. Ma). Abbreviations: INSL3, insulin-like peptide 3; LGR7/8, leucine-rich repeat-containing G-protein-coupled receptor-7/8 (also known as RXFP1/2).

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.03.076

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et al., 1993) and the human RLN3 gene equivalent in mouse and rat is designated Rln3 (Bathgate et al., 2002; Burazin et al., 2002). Here, ‘relaxin’ is used to refer to the peptide product of RLN2 (Rln1), the major bioactive form, while ‘relaxin-3’ refers to the peptide product of RLN3 (Rln3). It is now known that the RLN3 gene/peptide represent the ancestral form of the relaxin peptides (Wilkinson et al., 2005); and importantly, both relaxin and relaxin-3 mRNAs are expressed in brain (Osheroff and Ho, 1993; Bathgate et al., 2002; Burazin et al., 2002; Liu et al., 2003b). Extensive pharmacological studies have now shown that although relaxin binds and activates LGR7 and to a lesser extent LGR8, insulin-like peptide 3 (INSL3; or relaxinlike factor) binds and activates LGR8 at a higher affinity than relaxin, indicating that INSL3 is the cognate ligand for LGR8 (Kumagai et al., 2002; Bathgate et al., 2005; Scott et al., 2005). In fact, deletion of INSL3 (or Insl3) or LGR8 (or Lgr8), results in the same phenotype, cryptorchidism (Overbeek et al., 2001; Ivell and Bathgate, 2002; Kumagai et al., 2002; Bogatcheva et al., 2003; Ivell and Hartung, 2003) and similarly deletion of RLN1 (or rln1) or LGR7 (or Lgr7) results in the same phenotype of impaired nipple development and parturition (Zhou et al., 1999; KrajncFranken et al., 2004); providing convincing evidence for the native INSL3-LGR8 and relaxin-LGR7 pairings (Kamat et al., 2004). Furthermore, soon after the identification of relaxin-3, additional G-protein-coupled receptors (GPCR135 and -142) were identified as receptors for relaxin-3 that are insensitive to relaxin and most other members of the insulin/relaxin family (Liu et al., 2003a,b). So despite pharmacological activity of relaxin-3 at LGR7 in vitro and in vivo (Sudo et al., 2003; Bathgate et al., 2006b), relaxin-3 is believed to be the native ligand for GPCR135 in brain (Liu et al., 2003b; Sutton et al., 2004), while insulin-like peptide 5 (INSL5) is the preferred ligand for GPCR142 (Liu et al., 2005) and this receptor is not expressed in rat or mouse brain (Sutton et al., 2006). Prior to the identification of LGR7, central relaxin receptor localization was studied by radioligand autoradiography. Isotopically-labeled relaxin peptide was used to identify the distribution of relaxin binding sites and revealed a distinct distribution of putative relaxin receptors throughout cortical and sub-cortical areas of male and female rats (Osheroff et al., 1990; Osheroff and Phillips, 1991), including areas implicated in described autonomic and neuroendocrine physiological actions and distinct areas implicated in sensory and higher function. In line with the recent advances in the molecular characterization of LGR7, the current study describes the distribution of LGR7 mRNA throughout the rat brain detected using in situ hybridization histochemistry, compared with the distribution of [33P]-human relaxin binding sites, detected by autoradiography, providing strong evidence that LGR7 is indeed the preferred, high affinity receptor for relaxin in the rat brain. Preliminary reports of these findings have been published (Burazin et al., 2005; Ma et al., 2005; Bathgate et al., 2006b).

EXPERIMENTAL PROCEDURES Animals and tissue processing All animal experimental procedures were approved by the Howard Florey Institute Animal Ethics Committee and were performed in strict accordance with the guidelines of the National Health and Medical Research Council of Australia. All efforts were made to minimize the number of animals used in this study. Male Sprague– Dawley rats (270 –300 g; n⫽6) from the Animal Research Centre (Canning Vale, WA, Australia) were used and were housed individually with ad libitum access to pelleted food and water. Rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and decapitated and brains removed, frozen on dry ice, and stored at ⫺80 °C. Coronal and parasagittal sections (14 ␮m) throughout the rostro-caudal and medio-lateral extent of the brain were cut on a cryostat at ⫺16 °C and mounted on poly-L-lysinecoated glass slides for in situ hybridization histochemistry, or on gelatin/chrom-alum-coated glass slides for radioligand binding autoradiography. For in situ hybridization histochemistry, sections were dehydrated in ethanol (70 –100%), delipidated in chloroform for 10 min to help to decrease non-specific ‘myelin binding’ of oligonucleotides during hybridization, rinsed and stored in 100% ethanol at 4 °C.

In situ hybridization histochemistry Five oligonucleotides complementary to nucleotides 287–324, 340 –378, 685–723, 1533–1571, 1705–1743 of the LGR7 cDNA sequence (accession No. AY509976) were prepared (Geneworks, Adelaide, SA, Australia) and labeled with [35S]-dATP (PerkinElmer Life Sciences, Boston, MA, USA) to a specific activity of ⱖ1–2⫻109 d.p.m./␮g, as previously described (Burazin et al., 2002; Wisden and Morris, 2002). The sequence of each oligonucleotide was checked using the GenBank database for 100% homology to the target gene, and less than 70% homology with other mammalian genes. Labeled probes were diluted in hybridization buffer (1–5 pg/␮L per probe) consisting of 50% (v/v) formamide, 10% (v/v) dextran sulfate in 4⫻ SSC (1⫻ SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Dithiothreitol (200 mM) was added to the solution to minimize disulfide bridge formation and non-specific ‘hybridization.’ Brain sections were incubated for 16 h in hybridization buffer containing labeled probes at 42 °C. Specificity of hybridization was assessed in a 1:5 series of sections, by the addition of a 100-fold excess of unlabeled oligonucleotides to the hybridization buffer. In all experiments, specific hybridization was successfully displaced by this procedure. Labeled tissue sections were apposed to Kodak BioMax film (Integrated Sciences, Sydney, NSW, Australia) for 6 weeks. In addition, some sections were coated with nuclear emulsion (Ilford K5, diluted 1:1 with dH2O; Ilford Imaging, Melbourne, VIC, Australia) and exposed for 10 –12 weeks, prior to development, counterstained with 0.01% Thionin and analysis using bright- and dark-field microscopy.

Radioligand binding autoradiography [33P]-Human relaxin binding to brain sections was assessed as previously described (Osheroff et al., 1990; Tan et al., 1999). Coronal and sagittal sections (14 ␮m; near adjacent to those used for in situ hybridization; n⫽6) mounted on gelatin/chrom alumcoated glass microscope slides were pre-incubated for 30 min in 25 mM HEPES, 300 mM KCl, pH 7.2 containing 1 mM phenylmethanesulfonyl fluoride (Sigma-Aldrich, Castle Hill, NSW, Australia) at room temperature. Slides were then incubated in the same buffer plus 1% BSA (w/v), and 100 pM [33P]-B29 human relaxin (kindly supplied by Dr. R. Bathgate, Howard Florey Institute, Melbourne, VIC, Australia; Osheroff et al., 1990) at room temperature for 1 h. Non-specific binding of [33P]-relaxin was determined in the presence of 1 ␮M unlabeled

S. Ma et al. / Neuroscience 141 (2006) 329 –344 B29 human relaxin (kindly supplied by Dr. J. Wade, Howard Florey Institute). Following incubation, slides were washed in ice-cold buffer for 2⫻5 min and rinsed in ice-cold deionized water. Sections were allowed to air-dry overnight and were apposed to Kodak Biomax film (Eastman Kodak Company, New York, NY, USA) for 2 weeks.

Analysis of relative abundance of LGR7 mRNA and [33P]-relaxin binding sites Relative levels of hybridization and binding were assessed by visual inspection of X-ray film images and graded according to the scale of: (⫺) no hybridization, equivalent to general tissue/film background; (⫹) weak scattered hybridization or binding in the region; (⫹⫹) low, (⫹⫹⫹) moderate, or (⫹⫹⫹⫹) high level/density of hybridization/binding in a distinct nucleus/region; (⫹⫹⫹⫹⫹) intense level/density of hybridization/binding of a nucleus/region. For example, LGR7 mRNA hybridization was intense in the basolateral amygdala, high in the centromedial thalamic nucleus, moderate in the anterior pretectal nucleus and low in the arcuate hypothalamic nucleus (see Results). Appropriate stereotaxic atlases were used to identify brain structures (Paxinos and Watson, 1997).

Photography and image production Digital images from X-ray film were captured using MCID-M2 software (Imaging Research Inc., St. Catharine’s, Ontario, Canada) via a Sony XC-77 camera (Berthold Australia, Bundoora, VIC, Australia). Brightness and contrast levels of these images were adjusted and the composite plates were compiled and labeled using Adobe® Photoshop® 7.0 for Macintosh OSX (Adobe Systems Inc., San Jose, CA, USA). Digital micrographs of nuclear emulsion-dipped, counterstained sections were produced under bright- and dark-field illumination on a Nikon Microphot SA microscope (FSE, Melbourne, VIC, Australia) equipped with a Sony CCD camera, using MCID-M2 software. All micrographs were archived as high-resolution images in Adobe® Photoshop® 5.0

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(Adobe Systems Inc.) and after any required cropping, adjustment for contrast or removal of obvious artifacts; plates were compiled and labeled in PowerPoint (Microsoft Corporation, Mountain View, CA, USA). Schematics illustrating the distribution of LGR7 mRNA and [33P]-relaxin binding sites were created using Brain Maps: Structure of the Rat Brain software (Swanson, 1998, 1999) and Adobe® Photoshop® 7.0 for Macintosh OSX (Adobe Systems Inc.).

RESULTS The distribution of LGR7 mRNA throughout the rostrocaudal extent of the adult, male rat brain was determined by in situ hybridization histochemistry using multiple, specific oligonucleotide probes. In near adjacent sections, the distribution of putative LGR7 protein (binding sites) was assessed by autoradiography of [33P]-relaxin binding. Visual inspection of comparative X-ray film images revealed a good correlation between the broad, regional distributions of LGR7 mRNA and [33P]-relaxin binding sites in most areas, as illustrated in both parasagittal and coronal brain sections (Figs. 1 and 2). This relative distribution and abundance of LGR7 mRNA and putative protein was further documented on a schematic coronal map of rat brain (Fig. 3) and by semi-quantitative scoring of hybridization and binding densities (Table 1), respectively. The neuronal expression of LGR7 mRNA in these various brain areas was confirmed by high-resolution nuclear emulsion autoradiography of hybridized sections (Fig. 4). Further descriptions of these data are given below, along with brief details of the comparative topography of neurons and associated projections that contain the putative ligand for LGR7, relaxin or the related peptide, relaxin-3.

Fig. 1. Comparative distribution of LGR7 mRNA and [33P]-human relaxin binding sites in sagittal sections of adult rat brain. (A) Representative autoradiogram illustrating extensive LGR7 mRNA expression throughout layers 5 and 6b (and layer 1) of neocortex, in the anterior olfactory nucleus, CA3 field of hippocampus and the subiculum. (B) [33P]-Relaxin binding sites in a near-adjacent section are present in layers 1, 5, and 6b of NeoCx, the AOV, DG, SO and the Sub. The specificity of the hybridization and radioligand binding was demonstrated by the addition of a 100-fold excess of unlabeled probes to the hybridization buffer to reveal any non-specific hybridization (NSH) (A=) and the addition of 1 ␮M unlabeled relaxin peptide to the incubation buffer to detect levels of non-specific binding (NSB) (B=), respectively. Scale bar⫽2 mm.

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Abbreviations used in the figures ac ACB AH AOB AOD AOL AOM AON AOP AOV APir APTD Arc AUD AV BLA BMA BNST CA1-3 CEA Cl CL CM cp CPu CUN DC DCIC DG DLG DMH DpG DR DTg Ent EPd EPl f fi Gr Gl GP gVIIn Hb HDB IAM ic IC InG InWh IPN IPNdl IPNi IPNvl LD LDT LH LM LPO LS LVe MA ME mfb MG Mi

anterior commissure accumbens n anterior hypothalamic area accessory olfactory bulb anterior olfactory n, dorsal anterior olfactory n, lateral anterior olfactory n, medial anterior olfactory n anterior olfactory n, posterior anterior olfactory n, ventral amygdalopiriform transition area anterior pretectal n, dorsal arcuate hypothalamic n auditory cortex anteroventral thalamic n basolateral amygdala basomedial amygdala bed n of the stria terminalis CA1-3 fields of hippocampus central amygdala claustrum centrolateral thalamic n centromedial thalamic n cerebral peduncle caudate putamen cuneate n dorsal cochlear n dorsal cortex, inferior colliculus dentate gyrus of hippocampus dorsal lateral geniculate n dorsomedial hypothalamic n deep gray layer, superior colliculus dorsal raphe dorsal tegmental n entorhinal cortex dorsal endopiriform n external plexiform layer of olfactory bulb fornix fimbria of hippocampus granule cell layer of olfactory bulb glomerular layer of olfactory bulb globus pallidus genu of facial nerve habenula n n of horizontal limb of diagonal band interanteromedial thalamic n internal capsule inferior colliculus intermediate gray layer, superior colliculus intermediate white layer, superior colliculus interpeduncular n interpeduncular n, dorsolateral interpeduncular n, intermediate interpeduncular n, ventrolateral laterodorsal thalamic n laterodorsal tegmental n lateral hypothalamic area lateral mammillary n lateral preoptic n lateral septum lateral vestibular n medial preoptic area median eminence medial forebrain bundle medial geniculate n mitral cell layer of olfactory bulb

ml ML mlf MM MnPO MO MPN MS mt ND NeoCx NIc NId OB Op opt ox PAG pc PCL PG PIN Pir Pf PH PMD Pn PO PSV PV PVN RE RM RN RR Rt SC SCh scp SFO sm SN SNC SNR SO SPV st str Sub SuG SuM TRN TT VC VDB VIS VL VMH VP VPM VTA ZI 1-6b 4V *

medial lemniscus medial mammillary n, lateral medial longitudinal fasciculus medial mammillary n, medial median preoptic n motor cortex median preoptic n medial septum mammillothalamic tract n of Darkschewitsch neocortex nucleus incertus, compactus nucleus incertus, dissipata olfactory bulb optic nerve layer, superior colliculus optic tract optic chiasm periaqueductal gray posterior commissure paracentral thalamic n pontine n pineal gland piriform cortex parafascicular thalamic n posterior hypothalamic area premamillary n, dorsal part pontine reticular n posterior thalamic nuclear group principal sensory trigeminal n, ventrolateral paraventricular thalamic n paraventricular hypothalamic n reuniens thalamic n median raphe n red n retrorubral n reticular thalamic n superior colliculus suprachiasmatic n superior cerebellar peduncle subfornical organ stria medullaris of thalamus substantia nigra substantia nigra, compacta substantia nigra, reticulata supraoptic hypothalamic n spinal vestibular n stria terminalis superior thalamic radiation subiculum superficial gray layer, superior colliculus supramammillary n reticulotegmental n tenia tecta ventral cochlear n n of vertical limb of diagonal band visual cortex ventrolateral thalamic n ventromedial hypothalamic n ventral pallidum ventral posteromedial thalamic n ventral tegmental area zona incerta layer 1-6b cerebral cortex fourth ventricle tissue fold or hybridization/binding artifact

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Fig. 2. Comparative distribution of (A–I) LGR7 mRNA and (A=–I=) [33P]-human relaxin binding sites in coronal sections of adult rat brain. Non-specific hybridization levels were even and near-equivalent to film background (data not shown). (A⬙–I⬙) The specificity of radioligand binding is demonstrated by the addition of 1 ␮M unlabeled relaxin peptide to the incubation buffer. Scale bar⫽2.5 mm (A, B); 1 mm (C–I).

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Fig. 2. (Continued).

Regional and cellular distribution of LGR7 mRNA and [33P]-relaxin binding sites in adult rat brain Olfactory regions. In the olfactory bulb, low to moderate levels of LGR7 mRNA were observed in the mitral

and glomerular cell layers (Gl), with corresponding levels of [33P]-relaxin binding sites (Figs. 1A, 2A and 4A–A⬙). The granule cell layer (Gr) contained moderate levels of hybridization signal, whereas [33P]-relaxin binding sites in this

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Fig. 3. Schematic representation of the distributions of LGR7 mRNA expression (circles) and relaxin binding sites (shading) relative to that of relaxin (mRNA/IR; filled triangles) and relaxin-3 (mRNA; open triangles) in rat forebrain. Map and atlas plates adapted from Brain Maps: Structure of the Rat Brain software (Swanson, 1998, 1999). Relaxin and relaxin-3 mRNA distribution in rat brain adapted from Osheroff and Ho (1993) and Burazin et al. (2002), respectively, and S. Ma and T.C.D. Burazin, unpublished observations.

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Fig. 3. (Continued).

region were relatively low (Figs. 1A, 2A and 4A–A⬙). However, analysis of X-ray film and nuclear emulsion autora-

diograms from control sections, revealed a significant level of non-specific ‘hybridization’ of oligonucleotides to the

S. Ma et al. / Neuroscience 141 (2006) 329 –344 Table 1. Regional distribution of LGR7 mRNA and [33P]-human relaxin binding sites in rat braina Brain area

Rhinencephalon Anterior olfactory n. Olfactory bulb, glomerular layer Olfactory bulb, granule cell layer Olfactory bulb, mitral cell layer Telencephalon Accumbens n. Amygdala, basolateral Amygdala, lateral Caudate putamen Cerebral cortex, layer 1 Cerebral cortex, layer 2–4 motor, visual, auditory Cerebral cortex, layers 5–6 Cerebral cortex, layer 6b Claustrum/endopiriform n. Dentate gyrus (hilus) Entorhinal cortex Hippocampal CA1/2 Hippocampal CA3 Para/presubiculum Septum Subiculum Diencephalon Anterior hypothalamic n. Arcuate hypothalamic n. Bed n. of the stria terminalis Centrolateral thalamic n. Centromedial thalamic n. Habenula Medial mamillary n. Median preoptic hypothalamic n. Paracentral thalamic n. Paraventricular hypothalamic n. Reuniens thalamic n., medial Supramammillary n. Supraoptic n. Mesencephalon Anterior pretectal area Darkschewitsch n. Interpeduncular n. Substantia nigra, pars compacta Rhombencephalon Dorsal tegmental n. Locus coerulcus Median raphe Solitary tract n. Superior colliculus Circumventricular organs Organum vasculosum of the lamina terminalis Pineal gland Subfornical organ

LGR7 mRNA

[33P]-Relaxin binding sites

⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹

⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹

⫺ ⫹⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫺ ⫹ ⫺/⫹

⫺ ⫹⫹⫹⫹⫹ ⫹⫹⫹⫹⫹ ⫺ ⫹ ⫹

⫹⫹⫹⫹⫹ ⫹⫹⫹ ⫹ ⫺ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫹⫹ ⫺ ⫹⫹⫹

⫹⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫺ ⫹⫹⫹

⫺ ⫹⫹ ⫺ ⫹⫹⫹ ⫹⫹⫹⫹ ⫺ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹⫹ ⫹⫹

⫺ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹⫹⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹

⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹⫹ ⫹

⫹⫹ ⫺ ⫺ ⫹⫹ ⫹⫹

⫹⫹⫹ ⫺ ⫺ ⫹⫹ ⫹⫹⫹

⫹

⫹⫹

⫹⫹⫹⫹ ⫹⫹⫹⫹⫹

⫹⫹⫹⫹ ⫹⫹⫹⫹⫹

a

Relative level/density of LGR7 mRNA and [33P]-relaxin binding; ⫺, not detectable; ⫹, weak; ⫹⫹, low; ⫹⫹⫹, moderate; ⫹⫹⫹⫹, high; ⫹⫹⫹⫹⫹, intense.

compact granule cell layer (data not shown; see below). Therefore, the apparent levels of LGR7 mRNA-associated

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hybridization signal in the granule cell layer may appear in excess of the authentic level, which could be more in line with the low levels of relaxin binding sites observed. Moderate to high levels of LGR7 expression and relaxin binding sites were also observed in neurons of the claustrum and anterior olfactory nuclei/cortex (Figs. 1A, 2B and 4B–B⬙), an area that contains relaxin-producing cells (Osheroff and Ho, 1993; Burazin et al., 2001; Ma et al., 2005). Cerebral cortex. The neocortex contained high to very high levels of LGR7 mRNA associated with neurons in specific layers (Figs. 1A, 2B–H). Intense hybridization was present in presumably pyramidal cells throughout layer 5 and in a distinct group of cells in layer 6b, with lower levels associated with a smaller number of neurons in layer 1 (Figs. 1A, 2C–H and 4D–F; Table 1). Notably, low to undetectable levels of LGR7 mRNA were observed in primary olfactory (or piriform) cortex (Fig. 2C–E), an area known to contain a high concentration of relaxin-positive neurons in layers 2/3 (Osheroff and Ho, 1993; Burazin et al., 2001; Ma et al., 2005). A corresponding pattern of low to very high levels of [33P]-relaxin binding sites was observed throughout layers 1, 5 and 6b in the orbitofrontal, prefrontal, cingulate, insular, motor, parietal, somatosensory, auditory, visual, retrosplenial and rhinal cortices (Figs. 1B and 2B=–G). Only very low levels of binding sites were observed in layers 2– 4 throughout the entire neocortex, including primary and secondary motor, auditory, and visual cortices, suggesting in combination with the LGR7 mRNA distribution that very few binding proteins are located away from the neuronal soma located in the adjacent layers (see Fig. 3C–P). Relaxin mRNA has been detected in cells located within the somatosensory and motor cortex, although the precise layer was not stipulated (Osheroff and Ho, 1993) and moderate levels of relaxin mRNApositive cells are readily detected in the orbitofrontal cortex (Burazin et al., 2001). Hippocampal formation and amygdala complex. In the hippocampal formation, moderate levels of LGR7 mRNA were detected in the compact pyramidal cell layer of the CA3 (CA4) region, while low to undetectable levels were present in CA1/2 and in the granule cell layer of the dentate gyrus, particularly in the dorsoanterior hippocampus (Figs. 1A and 2D–E; Table 1). Moderate to high levels of LGR7 mRNA (and [33P]-relaxin binding sites) were observed in the dorsal subiculum (Figs. 1A, B and 2H, H=), whereas levels were relatively low in the entorhinal cortex. With the resolution afforded by X-ray film images, [33P]relaxin binding sites in the anterior hippocampus appeared enriched in the inner regions (hilus) of the dentate gyrus and little or no detectable binding was observed throughout areas CA1-3 (Figs. 1B and 2D=–E=). In the posterior hippocampus at the level of the midbrain, binding sites also appeared enriched in the dorsal compared with the ventral hilus of the dentate gyrus (Fig. 2G=–H=). High to intense levels of LGR7 mRNA were detected in the basolateral complex of the amygdala (Figs. 2D and 4F–F⬙; Table 1), with a corresponding abundance of [33P]relaxin binding sites that were clearly observed as a dis-

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Fig. 4. Regional and cellular localization of LGR7 mRNA in various areas of rat brain determined by in situ hybridization histochemistry. Nuclearemulsion autoradiograms confirm that LGR7 mRNA is associated with individual neurons. High densities of LGR7 mRNA-associated silver grains were observed over cells in (A–A⬙) the OB in the EPl and adjacent to olfactory glomeruli (inset); (B=–B⬙) the AOV; (C–E) the NeoCx in layer 5 (C=–C⬙), layer 6b (D–D=), and layer 1 (E); the BLA (F–F⬙); and (G–G⬙) the SFO. Scale bar⫽0.3 mm (A, B, C, F); 60 ␮m (A=, A⬙, B=, C=, D, E, F=, G, G=); 20 ␮m (B⬙, C⬙, D=, F⬙, G⬙, insets).

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tinct teardrop-shape comprising the lateral and basolateral subregions (Fig. 2D=). All remaining regions of the amygdala including the central and medial nuclei were essentially negative for LGR7 mRNA and [33P]-relaxin binding sites. In previous studies, relaxin mRNA has not been detected in cells within the amygdala, although it is expressed by neurons in cortical areas that innervate the basolateral amygdala (Osheroff and Ho, 1993; Ma et al., 2005). Hypothalamus. Moderate to high levels of LGR7 mRNA expression and corresponding levels of [33P]-relaxin binding sites were observed in the paraventricular and supraoptic hypothalamic nuclei (Fig. 2C, C=). Low to moderate levels of mRNA expression and binding sites were observed in the median preoptic nucleus (not shown), dorsolateral zones of the arcuate nucleus (Fig. 2D=) and medial and lateral regions of the supramammillary nucleus in the posterior hypothalamus (Fig. 2E, E=; Table 1). Neurons within the arcuate nucleus contain relatively high levels of relaxin mRNA and immunoreactivity (Ma et al., 2005). Thalamus. Moderate to high densities of LGR7 mRNA were observed in the paraventricular and centromedial nuclei of the anterior thalamus, with lower levels in the paracentral and centrolateral nuclei and a matching distribution pattern of [33P]-relaxin binding sites (Fig. 2C–D=). Low-moderate levels of LGR7 mRNA and [33P]-relaxin binding sites, diffuse and scattered in appearance, were observed in the medial reuniens nucleus (Fig. 2C) and in the posterior thalamus, possibly associated with the dorsal anterior pretectal nuclei (Fig. 2E–H=; Table 1). Circumventricular organs. Very high levels of LGR7 mRNA and corresponding densities of specific [33P]relaxin binding sites were observed in the subfornical organ in the external layers of the nucleus, which form an O-shape (Figs. 2C, C= and 4G, G⬙). Moderate-high levels of LGR7 mRNA and relaxin binding sites were also detected in the pineal gland (Fig. 3P), and low levels were detected in the organum vasculosum of the lamina terminalis (Table 1). Midbrain, pons and medulla. In the midbrain, intense levels of LGR7 mRNA and [33P]-relaxin binding sites were observed in the dorsolateral and ventrolateral regions of the interpeduncular nucleus and low levels of both LGR7 mRNA and [33P]-relaxin binding were observed throughout the superficial and deep layers of the superior colliculus and in the substantia nigra, pars compacta (Fig. 2G, G= and 2H, H=; Table 1). A moderate to high level of LGR7 mRNA and binding sites was observed within a midline structure tentatively identified from X-ray film images as the nucleus of Darkschewitsch (Osheroff and Phillips, 1991). High levels of LGR7 mRNA were detected in the dorsal tegmental nucleus of the pons along with corresponding levels of [33P]-relaxin binding sites (Fig. 2I, I=). Notably, the adjacent midline region known as the nucleus incertus (or nucleus O) is known to contain a large number of relaxin-3 producing neurons (Burazin et al., 2002; Liu et

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al., 2003b; Tanaka et al., 2005) that send projections to the dorsal tegmental nucleus and a range of other forebrain targets in the rat (Goto et al., 2001; Olucha-Bordonau et al., 2003; Tanaka et al., 2005). Most other regions and nuclei of the pons and medulla were negative for transcripts and binding sites, although expression was observed in sections through the nucleus of the solitary tract (Table 1). Brain regions lacking LGR7 mRNA and relaxin binding sites. Several major brain regions/nuclei, which are known to express numerous other neurotransmitter and peptidemodulator receptors, were negative for LGR7 transcripts and relaxin binding sites, including the nucleus accumbens, caudate putamen, septum, bed nucleus of the stria terminalis, lateral and anterior hypothalamus, the sensory thalamus and the habenula (Fig. 2C, C=–E, E=; Table 1). In the hindbrain, the locus coeruleus and raphe nuclei were negative for specific labeling; and the cerebellum was also negative for specific labeling throughout its rostrocaudal extent, despite an apparent hybridization signal in some sections, which was presumably non-specific accumulation of labeled oligonucleotide probes within densely-packed granule cells (Fig. 2I; data not shown).

DISCUSSION Early studies investigating the distribution of putative relaxin receptors in rat brain examined the localization of binding sites for radioactively-labeled relaxin peptide by autoradiography (Osheroff and Phillips, 1991). This method presumably revealed all high affinity binding sites for relaxin without providing information on the identity or characteristics of the receptor(s), which were unknown at the time. Considerable evidence has now demonstrated that LGR7 fulfils all the requirements of an authentic relaxin receptor. It binds relaxin (Hsu et al., 2002) (and the structurally-related relaxin-3 (Sudo et al., 2003)) with high affinity and is expressed in the late pregnant reproductive tract and other tissues known to be involved in relaxin physiology. Furthermore, the soluble ectodomain of the receptor when injected into late pregnant mice can delay parturition (Hsu et al., 2002), an effect that was also observed following neutralization of central relaxin by i.c.v. administration of a monoclonal antibody against the peptide in pregnant rats (Summerlee et al., 1998b). Furthermore the phenotype of Lgr7(⫺/⫺) and rln(⫺/⫺) mice is largely identical (Zhao et al., 1999; Krajnc-Franken et al., 2004; see above) and while it was initially reported that LGR8 was also a putative receptor for relaxin (Hsu et al., 2002), a range of subsequent studies have determined that INSL3 is the preferred endogenous ligand for LGR8 (Kumagai et al., 2002; Bathgate et al., 2005; Scott et al., 2005), and LGR8 is expressed in a contrasting pattern to that for LGR7 in rat brain (Shen et al., 2005). Thus, the current study examined whether the distribution of LGR7 mRNA correlated with that of [33P]-relaxin binding sites.

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Localization of LGR7 in brain: anatomical considerations These studies have identified the extensive, topographical distribution of LGR7 mRNA throughout the adult, male rat brain, with expression detected in specific regions/nuclei of the olfactory bulb, neocortex, limbic forebrain, hypothalamus, thalamus, midbrain, pons and medulla; and highest densities of receptor transcripts observed in the anterior olfactory nuclei/cortex, deep layers of the cingulate, somatosensory and motor cortices, basolateral amygdala, paraventricular and supraoptic nuclei, several intralaminar thalamic nuclei, interpeduncular and dorsal tegmental nuclei, and in the subfornical organ. All these areas contained a correspondingly abundant and regionally restricted level of [33P]-relaxin binding sites. These data suggest that the LGR7 protein is predominantly expressed on neuronal soma, and is not extensively transported axonally and expressed as a ‘presynaptic’ receptor on nerve terminals in adjacent or distant regions. Thus LGR7 is positioned to act as a postsynaptic receptor for remotely-produced and/or synaptically-released relaxin, or as an autoreceptor for locally-produced relaxin. In all the material examined, the hippocampus was one area where this strict correlation was not so clearly observed. Low and moderate levels of LGR7 mRNA were observed by both X-ray film and highresolution nuclear emulsion autoradiography in the compact and more dispersed pyramidal cell layers of CA1 and CA3 respectively, throughout the rostro-dorsal to caudoventral extent of the hippocampus. Relaxin binding sites appeared enriched in the inner polymorph or hilar region of the dentate gyrus (i.e. near adjacent to the broad band of CA3 cells), which is not inconsistent with LGR7 protein expression by these pyramidal neurons at the level of the soma or their dendrites. One alternative explanation is that receptors present in the dentate hilus are wholly or partly expressed by intrinsic cells within the area, an idea supported by putative LGR7-immunostaining of hilar interneurons in the rat (Burazin et al., 2005) and LGR7 gene expression in such cells in the mouse brain (Piccenna et al., 2005), but not clearly supported by the current in situ hybridization studies in rat. Thus, further studies are required to confirm which LGR7 mRNA-expressing cells are the source(s) of these receptor proteins. Endogenous source(s) of ligand for LGR7: central relaxin and relaxin-3 neurons Another important aspect of putative LGR7 signaling in deeper brain structures is the source of ligand to activate these receptors. In this regard, early and more recent anatomical studies have revealed that the distributions in rat brain of the two putative ligands for LGR7, relaxin and relaxin-3 (Osheroff and Ho, 1993; Burazin et al., 2001, 2002) are more restricted than that of putative relaxin receptors (Osheroff and Phillips, 1991) suggesting the existence of neuronal projections containing relaxin peptide(s) innervating multiple target regions, in a way predicted/expected for a ‘legitimate’ neuropeptide system.

Using in situ hybridization histochemistry, relaxin mRNA is readily detected in neurons in several areas of rat forebrain, including anterior olfactory nuclei/cortex, frontal/ orbital and olfactory cortices, and also in lower abundance in hypothalamus, and neocortex and hippocampus (Osheroff and Ho, 1993; Burazin et al., 2001). It is known that neurons of the rat olfactory cortex and anterior olfactory nuclei have strong reciprocal connections with the olfactory bulb (Luskin and Price, 1983; Haberly, 2001; Brunjes et al., 2005), and so these regions may be potential sources of relaxin peptide for LGR7 present in the olfactory bulb. Moreover, pyramidal neurons of the olfactory/(piriform) cortex send strong projections to various forebrain regions (Luskin and Price, 1983; Haberly, 2001; Brunjes et al., 2005), including insular areas of the neocortex, amygdala, entorhinal cortex and subiculum, mediodorsal thalamus and hypothalamus, where LGR7 mRNA and corresponding relaxin binding sites were observed. Notably, in preliminary studies of the possible regulation of relaxin expression in the piriform cortex of rats, we observed an increase in relaxin mRNA three hours after acute pentylenetetrazole-induced seizures (S. Ma and A. L. Gundlach, unpublished observations), suggesting that relaxin expression in these cells can be regulated by (patho)physiological stimuli. Similarly, neurons in the arcuate nucleus that produce relaxin may activate LGR7 on adjacent cells or on cells in more distant regions of the hypothalamus, the limbic system or brainstem, autonomic nuclei. Under certain conditions relaxin may also be released onto the pituitary gland (see below). In contrast, relaxin-3 mRNA is highly expressed in neurons in the pontine nucleus incertus in mouse (Bathgate et al., 2002) and rat brain (Burazin et al., 2002; Liu et al., 2003b) and relaxin-3 immunoreactive nerve fibers have been found to be broadly distributed within rat forebrain, using both a N-terminal-directed mouse monoclonal antibody (Tanaka et al., 2005) and a C-peptide-directed rabbit polyclonal antisera (Banerjee et al., 2005). However, the distribution of relaxin-3-containing nerve fibers in rat brain correlates better with the distribution of GPCR135 mRNApositive cells and specific binding sites for a chimeric relaxin-3 peptide selective for GPCR135 (Sutton et al., 2004). There are nonetheless, several brain regions such as the paraventricular and supraoptic hypothalamic nuclei and the paraventricular and other midline thalamic nuclei that contain GPCR135 mRNA- and LGR7 mRNA-positive cells; and as relaxin-3 can bind and activate LGR7 in vitro and in vivo (Liu et al., 2005; Bathgate et al., 2006b), relaxin-3 may act at both receptors under certain physiological conditions. This may depend on the local concentration of the peptide, as human relaxin-3 preferentially activates human GPCR135 cf. LGR7 (EC50 ⬍1 nM vs ⬃20 nM; Liu et al., 2003b; Sudo et al., 2003). Further studies are required to clarify this issue. Possible functional roles for relaxin-LGR7 signaling in rat brain Before the identification of its native receptor, the actions of relaxin ‘on and within’ the brain were investigated by

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several laboratories. Peripheral and central administration of relaxin in rats altered central hemodynamic control by induction of drinking, via actions on circumventricular and hypothalamic nuclei such as the organum vasculosum of the lamina terminalis, subfornical organ and supraoptic and paraventricular nuclei (Geddes and Summerlee, 1995; Thornton and Fitzsimons, 1995; McKinley et al., 1997; Summerlee et al., 1998a; Sinnayah et al., 1999), effects related to cardiovascular changes that occur during pregnancy. Relaxin also had effects on oxytocin and vasopressin secretion, reflected by an elevation of plasma vasopressin and oxytocin concentration and increased systolic and diastolic blood pressure in pregnant and lactating rats after acute systemic administration (Summerlee et al., 1984; Parry et al., 1994). Central (i.c.v.) and systemic administration of relaxin also significantly elevated Fos-like immunoreactivity in oxytocin neurons in the supraoptic nuclei, magnocellular cells of the rostral and caudal paraventricular nuclei, and dorsal, organum vasculosum of the lamina terminalis (Heine et al., 1997; McKinley et al., 1997), via a combination of direct and neural pathway activation effects (McKinley et al., 2004). Furthermore, a relaxin monoclonal antibody administrated i.v. disrupted the birthing process, but not its timing; but when administered centrally, disrupted the timing of parturition (Summerlee et al., 1998b), effects attributed to actions at receptor-rich circumventricular organs and hypothalamic nuclei that lie along the border of ventricles and have a compromised blood– brain barrier, allowing direct exposure to blood- or cerebrospinal fluid-borne peptides and hormones (Gross, 1992). Somatosensory processing associated with memory, emotion and integrated behavior. In the olfactory bulb, low to moderate levels of LGR7 mRNA and relaxin binding sites were found in the granule cell and (peri-)glomerular layers. LGR7-null mice with a LacZ cassette in place of the deleted sequence, also display high levels of associated ␤-galactosidase staining in cells of the peri-glomerular layer of the olfactory bulb (Piccenna et al., 2005). LGR7 mRNA and relaxin binding sites were also observed in the anterior olfactory nuclei (or cortex), where relaxin mRNA and immunoreactivity are expressed (Osheroff and Ho, 1993; Burazin et al., 2001; Ma et al., 2005). Notably, despite the presence of relaxin in the associated primary olfactory (or piriform) cortex, no LGR7 mRNA or binding sites were observed in this region. The olfactory bulb and cortex are thought to mediate processing of olfactory information via strong connections with high-order association areas involved with learning, emotion, and other behavioral functions (Johnson et al., 2000). For example, the strong reciprocal connections between the piriform cortex and the basolateral amygdala, where LGR7 is abundant, are well established as a functional neural circuit involved in olfactory learning (Schoenbaum et al., 1998), suggesting, along with other evidence (see below), a role for LGR7 in this type of cognition. Several other brain regions involved in higher-order processes also express moderate to high densities of

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LGR7 mRNA and relaxin binding sites. In the amygdala, dense LGR7 mRNA and relaxin binding sites were observed in the basolateral complex, consisting of the lateral, basal, and accessory basal nuclei. It is well established that the amygdala is involved in emotional processes associated with sensory information and the formation of fear memory (Hamann et al., 1999; Cardinal et al., 2002; McGaugh et al., 2002; McGaugh, 2004) and we have recently reported the ability of exogenous relaxin administration into the basolateral amygdala to impair fear-related memory consolidation, in an aversively-motivated inhibitory avoidance task in rats (Ma et al., 2005). Relaxin-induced impairment of memory consolidation suggests that relaxin inhibits overall BLA neuronal activity through LGR7. This is consistent with preliminary findings that indicate that relaxin (ⱕ100 nM) inhibits forskolin-induced adenylate cyclase activity/cAMP accumulation in rat cortical membranes and inhibits the activity of rat BLA pyramidal neurons recorded in vitro (Ma et al., 2003). However, further studies are required to provide better insights into these cellular and physiological functions of relaxin. It will also be important to determine whether and how actions of relaxin on memory are related to actions in regulating associated neuroendocrine, autonomic and reproductive processes and to determine the role of relaxin in other LGR7-rich regions implicated in animal learning and memory including the neocortex, intralaminar and pretectal thalamic nuclei, hippocampus/dentate gyrus and supramammillary nucleus. Neuroendocrine and autonomic regulation. Multiple hypothalamic nuclei are known to play a primary role in the mediation and regulation of neuroendocrine function in mammals; and the activity of different hypothalamic neuron populations is altered by a range of physiological states and stimuli such as pregnancy and lactation, feeding and metabolism and changes in body temperature and serum osmolarity, and associated changes in various hormones and/or neuropeptide and cytokine regulatory factors. Importantly, LGR7 mRNA and relaxin binding sites are observed in various hypothalamic nuclei, suggesting a role of relaxin in modifying the neuroendocrine processes associated with these nuclei, such as oxytocin and vasopressin release in the paraventricular and supraoptic nuclei and the secretion of a range of peptides that influence feeding, metabolism, growth and reproduction in the arcuate nucleus and the pituitary gland. Preliminary studies in our laboratory have identified LGR7-like immunoreactivity enriched in oxytocin cells of the paraventricular and supraoptic hypothalamic nucleus (Burazin et al., 2005), consistent with direct effects of relaxin on oxytocin neuron activity and oxytocin release (Way and Leng, 1992). Relaxin also inhibited oxytocin and vasopressin release from isolated neural lobes of the male rat pituitary under basal conditions, but potentiated secretion when nerve endings of the neurohypophysis were depolarized (Dayanithi et al., 1987), suggesting the presence of functional relaxin receptors (LGR7) on magnocellular nerve efferents (perhaps unlike other deeper brain areas).

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In the current and previous studies (Ma et al., 2005), LGR7 mRNA and relaxin mRNA/immunoreactivity were also observed in neuronal cell bodies of the arcuate nucleus, suggesting that LGR7 functions as a local feedback regulator of relaxin synthesis and/or release into the hypothalamic–pituitary portal circulation to influence the release of hormones from the anterior pituitary. Studies of the co-localization of relaxin and LGR7 in arcuate neurons are now required to validate this possibility. LGR7 mRNA and relaxin binding sites have not been reported in the rat pituitary gland and their presence was not directly examined here, but early studies reported that relaxin increased intracellular cAMP generation in cultured female and male rat anterior pituitary cells (Cronin and Malaska, 1989) and increased prolactin secretion from cultured anterior pituitary cells from cycling female rats (Sortino et al., 1989). Furthermore, a recent study in LGR7-null/LacZ-positive mice found high levels of associated ␤-galactosidase activity/(staining) in the pituitary gland of pregnant mice, with no staining in non-pregnant and male mice (Krajnc-Franken et al., 2004). In summary, the widespread distribution of LGR7 mRNA and relaxin binding sites in cortical, thalamic, limbic and hypothalamic regions of the rat brain strongly supports a role for relaxin-LGR7 signaling in higher-order somatosensory processing associated with memory, emotion, arousal and other behaviors. The richest sources of central relaxin in rat forebrain appear to be the anterior olfactory nuclei and the piriform and orbitofrontal cortices (and possibly other cortical areas) as well as the arcuate nucleus of the hypothalamus; and LGR7 are presumably responsive to altered levels of relaxin peptide in these areas or in the circulation, under different physiological conditions.

CONCLUSION The current study revealed a distinct distribution of LGR7 mRNA in rat brain that correlates closely with that of [33P]relaxin binding sites, strongly suggesting that these sites represent the presence of functional LGR7 protein. These findings further support separate pharmacological and molecular genetic evidence that relaxin is the cognate ligand for LGR7, and although there is an apparent mismatch between the restricted versus widespread distribution of relaxin and LGR7 protein, respectively, it is possible that relaxin is synthesized in particular neurons and axonally transported to nerve terminals in distant regions for secretion onto cells that express LGR7 or that relaxin activates LGR7 more via ‘volume transmission.’ While further studies are required to clarify the precise nature of the ligandreceptor topography and interaction, the distribution of LGR7 mRNA/protein strongly suggests functions for LGR7 signaling in higher-order processes involved in somatosensory, autonomic and neurohumoral pathways, which warrants further investigation. Acknowledgments—This work was supported by an Institute Block Grant (983001) to the Howard Florey Institute from the National Health and Medical Research Council (NHMRC) Australia and a grant from the Brain Foundation Australia (A.L.G.).

During these studies S.M. was the recipient of an NHMRC (Australia) Dora Lush Biomedical Postgraduate Scholarship and T.C.D.B. was an NHMRC Peter Doherty Fellow. The authors wish to thank Dr. Ross Bathgate and Dr. John Wade for the supply of radiolabeled and unlabeled peptides and the Rebecca L. Cooper Medical Research Foundation and BAS Medical, San Mateo CA, USA for their support.

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(Accepted 23 March 2006) (Available online 24 May 2006)