Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain

Distribution of Relaxin-3 and RXFP3 WithinArousal, Stress, Affective, and CognitiveCircuits of Mouse Brain

Craig M. Smith,1 Pei-Juan Shen,1 Avantika Banerjee,1 Pascal Bonaventure,2 Sherie Ma,1

Ross A.D. Bathgate,1 Steven W. Sutton,2 and Andrew L. Gundlach1*1Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia2Neuroscience Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, San Diego, California 92121, USA

ABSTRACTRelaxin-3 (RLN3) and its native receptor, relaxin family

peptide 3 receptor (RXFP3), constitute a newly identi-

fied neuropeptide system enriched in mammalian brain.

The distribution of RLN3/RXFP3 networks in rat brain

and recent experimental studies suggest a role for this

system in modulation of arousal, stress, metabolism,

and cognition. In order to facilitate exploration of the

biology of RLN3/RXFP3 in complementary murine mod-

els, this study mapped the neuroanatomical distribution

of the RLN3/RXFP3 system in mouse brain. Adult, male

wildtype and RLN3 knock-out (KO)/LacZ knock-in (KI)

mice were used to map the central distribution of RLN3

gene expression and RLN3-like immunoreactivity (-LI).

The distribution of RXFP3 mRNA and protein was deter-

mined using [35S]-oligonucleotide probes and a radiola-

beled RXFP3-selective agonist ([125I]-R3/I5), respectively.

High densities of neurons expressing RLN3 mRNA,

RLN3-associated b-galactosidase activity and RLN3-LI

were detected in the nucleus incertus (or nucleus O),

while smaller populations of positive neurons were

observed in the pontine raphe, the periaqueductal gray

and a region adjacent to the lateral substantia nigra.

RLN3-LI was observed in nerve fibers/terminals in nu-

cleus incertus and broadly throughout the pons, mid-

brain, hypothalamus, thalamus, septum, hippocampus,

and neocortex, but was absent in RLN3 KO/LacZ KI

mice. This RLN3 neural network overlapped the regional

distribution of RXFP3 mRNA and [125I]-R3/I5 binding

sites in wildtype and RLN3 KO/LacZ KI mice. These

findings provide further evidence for the conserved na-

ture of RLN3/RXFP3 systems in mammalian brain and

the ability of RLN3/RXFP3 signaling to modulate ‘‘be-

havioral state’’ and an array of circuits involved in

arousal, stress responses, affective state, and cognition.

J. Comp. Neurol. 518:4016–4045, 2010.

VC 2010 Wiley-Liss, Inc.

INDEXING TERMS: amygdala; arousal; nucleus incertus; relaxin-3; RXFP3 (GPCR135); septohippocampal system; stress

Relaxin-3 (RLN3) is a newly identified member of the

relaxin/insulin-like family of peptides (Bathgate et al.,

2002) and is highly conserved across a range of species

from fish to mammals (Wilkinson et al., 2005). Identified

by our laboratory via its sequence homology to human

RLN1 and -2 (Bathgate et al., 2002), RLN3 has an insulin-

like structure, with 51 amino acid residues arranged into

two separate alpha helix chains, joined by two disulfide

bonds (Rosengren et al., 2006). RLN3 expression is

largely restricted to the brain (Bathgate et al., 2002), and

in the mouse and rat, RLN3 mRNA is highly enriched in

neurons of the midline, pontine nucleus incertus (NI) or

nucleus O (Bathgate et al., 2002; Burazin et al., 2002; Liu

et al., 2003b). In more recent studies in the rat, popula-

tions of RLN3 mRNA-positive cells were also detected in

the pontine raphe nucleus, the anterior, lateral, and

Additional Supporting Information may be found in the online versionof this article.

Grant sponsor: National Health and Medical Research Council ofAustralia; Grant numbers: 327404 (to C.M.S.), 520299 (to S.M.), 277609,509246 (to A.L.G.); Grant sponsors: Collaborative Research Agreementwith Johnson & Johnson PR&D, LLC, San Diego, CA, Howard FloreyBiomedical Foundation USA (to A.L.G.), The University of Melbourne –Melbourne (Postgraduate) Research Scholarship (to A.B.), ANZ TrusteesMedical Research & Technology (Victoria, Australia), Perpetual Trustees (toA.L.G., S.M.).

*CORRESPONDENCE TO: Andrew L. Gundlach, Florey NeuroscienceInstitutes, The University of Melbourne, Victoria 3010, Australia. E-mail:[email protected]

VC 2010 Wiley-Liss, Inc.

Received December 22, 2009; Revised April 29, 2010; Accepted June 1,2010

DOI 10.1002/cne.22442

Published online June 17, 2010 in Wiley InterScience (www.interscience.wiley.com)

4016 The Journal of Comparative Neurology | Research in Systems Neuroscience 518:4016–4045 (2010)

RESEARCH ARTICLE

ventrolateral periaqueductal gray, and in an area dorsal

to the lateral substantia nigra (Tanaka et al., 2005; Ma

et al., 2007).

Interest in the connectivity and neurochemistry of the

NI was raised by the discovery that NI neurons expressed

high levels of corticotrophin-releasing factor receptor-1

Abbreviations

n. Nucleusaca Anterior commissure, anteriorAcb Accumbens n.AD Anterodorsal thalamic n.AHi Amygdalohippocampal areaalv Alveus of the hippocampusAOM Anterior olfactory n., medialApir Amygdalopiriform transition areaAq AqueductAStr Amygdalostriatal transition areaAuC Auditory cortexB Barrington’s n.BLA Basolateral amygdalaBMA Basomedial amygdalaBNST Bed n. stria terminalisCA1-3 CA1-3 fields of hippocampusCB Cerebellumcc Corpus callosumCe(M) Central amygdala (medial part)CG Central grayCg(1/2) Cingulate cortex (area 1/2)Cl ClaustrumCM Centromedial thalamic n.cp Cerebral peduncle, basalCPu Caudate putamenD3V Dorsal 3rd ventricleDCIC Dorsal cortex, inferior colliculusDEn Dorsal endopiriform n.df Dorsal fornixDG Dentate gyrus hippocampusDLG Dorsal lateral geniculate n.DLPAG Dorsal lateral periaqueductal grayDMH Dorsomedial hypothalamic n.DMPAG Dorsal medial periaqueductal grayDpG Deep layer of the superior colliculusDR Dorsal raphe n.dSN Region dorsal to substantia nigraDT Dorsal terminal n. of the accessory optic tractDTg Dorsal tegmental n.DTT Dorsal tenia tectaec External capsulef Fornixfmj Forceps major corpus callosumGrO Granule cell layer olfactory bulbGrDG Granular layer, dentate gyrusgVIIn Genu of facial nerveHb HabenulaHDB N. horizontal limb diagonal bandHip Hippocampusic Internal capsuleIC Inferior colliculusIGL Intergeniculate leafletInG Intermediate gray layer of the superior colliculusInWh Intermediate white layer of the superior colliculusIO Inferior oliveIPN Interpeduncular n.IPDL Interpeduncular n., dorsolateralIPDM Interpeduncular n., dorsomedialIPL Interpeduncular n., lateralLa Lateral amygdalalat LateralLC Locus coeruleusLDTg Laterodorsal tegmental n.LEnt Lateral entorhinal cortexLH Lateral hypothalamic areaLM Lateral mammillary n.LPAG Lateral periaqueductal grayLPG Lateral parabrachial n.LPO Lateral preoptic area hypothalamusLPT Lateral posterior thalamic n.LS Lateral septumLSD Lateral septal n., dorsalLT Lateral terminal n. of the accessory optic tractLV Lateral ventricleM1 Primary motor cortexM2 Secondary motor cortexMe5 Mesencephalic trigeminal n.

MeA Medial amygdalaMEnt Medial entorhinal cortexmfb Medial forebrain bundleMGV Medial geniculate n., ventralml Medial lemniscusm/l Midlinemlf Medial longitudinal fasciculusMnR Median rapheMO Medial orbital cortexMol Molecular layer of the dentate gyrusMS Medial septummt Mammillothalamic tractMVePC Medial vestibular n. parvicellular partNI Nucleus incertusOp Optic nerve layer, superior colliculusopt Optic tractOr Oriens layer, hippocampusPAG Periaqueductal graypc Posterior commissurePe Periventricular hypothalamic n.PH Posterior hypothalamic areaPIL Posterior intralaminar thalamic n.Pir Piriform cortexpm Principal mammillary tractPnC Pontine reticular n., caudalPnR Pontine raphe n.PoDG Polymorph layer dentate gyrusPP Peripeduncular n.PPT Posterior pretectal n.PPTg Pedunculopontine tegmental n.Pr Prepositus hypoglossal n.PrL Prelimbic cortexPVA Paraventricular thalamic n., anteriorPVN Paraventricular hypothalamic n.Py Pyramidal cell layer, hippocampusRC Raphe capRe Reuniens thalamic n.RLi Rostral linear rapheRLN3 Relaxin-3RMg Raphe magnus n.RS Retrosplenial cortexRSA Retrosplenial agranular cortexRSG Retrosplenial granular cortexRXFP3 Relaxin family peptide 3 receptorS SubiculumS1 Primary somatosensory cortexS2 Secondary somatosensory cortexSC Superior colliculusSCh Suprachiasmatic n.SFi Septofimbrial n.SG Supragenual n.SHi Septohippocampal n.SI Substantia innominataSN Substantia nigrasol Solitary tractSON Supraoptic n.SPF Subparafascicular thalamic n.st Stria terminalisSuG Superficial gray layer, superior colliculusSuM Supramammillary n.SuML Supramammillary n. lateral partSuMM Supramammillary n. medial partsumx Supramammillary decussationTS Triangular septal n.V1 Primary visual cortexV2 Secondary visual cortexVDB N. vertical limb diagonal bandVEn Ventral endopiriform cortexVIIn Facial nerveVLG Ventral lateral geniculate n.VLPAG Ventral lateral periaqueductal grayVMH Ventromedial hypothalamic n.VTA Ventral tegmental areavtgx Ventral tegmental decussation peduncleZI Zona incerta3V Third ventricle4V Fourth ventricle

Relaxin-3/RXFP3 distribution in mouse brain

The Journal of Comparative Neurology | Research in Systems Neuroscience 4017

(CRF1) mRNA and were activated following intracerebro-

ventricular (i.c.v.) injections of CRF (Potter et al., 1994;

Bittencourt and Sawchenko, 2000). Immunohistochemi-

cal studies reported that NI neurons in the rat express

the GABA-synthesizing enzyme, glutamate decarboxylase

(Olucha-Bordonau et al., 2003), and that the majority, if

not all, RLN3 neurons in the NI are GAD-positive (Ma

et al., 2007). Other peptides detected in the rat NI (or

equivalent) include cholecystokinin (Olucha-Bordonau

et al., 2003), galanin, dynorphin (Sutin and Jacobowitz,

1988), neurotensin (Jennes et al., 1982), and a ranaten-

sin-like peptide (probably neuromedin-B) (Chronwall

et al., 1985). Dense networks of RLN3-immunoreactive

nerve fibers have been described in rat brain (Tanaka

et al., 2005; Ma et al., 2007) and electron microscopy

studies indicate that RLN3 is translated in the rough

endoplasmic reticulum, packaged into dense-core

vesicles and trafficked to presynaptic sites in nerve termi-

nals (Tanaka et al., 2005), consistent with a role as a

transmitter/modulator.

Anterograde and retrograde tract-tracing studies in the

rat demonstrated that NI neurons have strong reciprocal

connections with the median raphe (or central superior)

and interpeduncular nuclei, forming a likely behavioral

control network (Goto et al., 2001; Olucha-Bordonau

et al., 2003) that uses GABA and/or a range of peptides

including RLN3 to influence various neural networks

involved in behavioral activation. A heavy innervation of

the septohippocampal system by the NI suggested an

involvement in the control of hippocampal theta rhythm—

a process implicated in memory and attentional states,

such as exploratory behavior and rapid eye movement

(REM) sleep (Buzsaki, 2005; Hasselmo, 2005; Vertes,

2005). In support of this view, electrical stimulation of

the NI in anesthetized rats induced hippocampal theta

rhythm, while electrolytic lesion or pharmacological

blockade of the NI disrupted hippocampal theta induced

by stimulation of the reticularis pontis oralis (Nunez et al.,

2006), a pontine region known to be an endogenous ini-

tiator of hippocampal theta rhythm that is strongly inter-

connected with the NI (Teruel-Marti et al., 2008).

Molecular and pharmacological studies have demon-

strated that the native RLN3 receptor is the relaxin family

peptide 3 receptor (RXFP3; Bathgate et al., 2006), which

was formally known as G-protein-coupled receptor 135

(GPCR135; Liu et al., 2003a,b; Sutton et al., 2006), and

somatostatin- and angiotensin-like peptide receptor

(SALPR; Matsumoto et al., 2000). Activation of RXFP3

stably expressed in Chinese hamster ovary CHO-K1 or

human embryonic kidney HEK-293 cells results in an inhi-

bition of cAMP accumulation via Gi/o-protein coupling

(Liu et al., 2003b; Ortinau et al., 2005) and activation of

extracellular signal-regulated kinase 1/2 in these cells

and the mouse septal neuronal cell line SN-56 (van der

Westhuizen et al., 2007), but the precise nature of endog-

enous RXFP3 signaling in neurons or particular neural net-

works is currently unclear.

The distribution of RXFP3 mRNA and binding sites in

rat brain has been mapped using in situ hybridization his-

tochemistry and radioligand autoradiography, respec-

tively, with the latter studies employing a radiolabeled

chimeric peptide ([125I]-RLN3 B-chain/INSL5 A-chain

(R3/I5)) that is highly selective for RXFP3 (Sutton et al.,

2004; Liu et al., 2005; Ma et al., 2007). The receptor dis-

tribution in forebrain largely overlapped the RLN3 inner-

vation, and together with experimental studies in rats,

suggests that RXFP3 signaling is involved in the regula-

tion of circuits involved in stress responses and feeding

behavior. Expression of CRF1 by RLN3 NI neurons is re-

sponsible for the elevation in c-fos/Fos expression in

these cells after i.c.v. injection of CRF (Bittencourt and

Sawchenko, 2000; Tanaka et al., 2005), and increased

RLN3 mRNA after water-immersion restraint stress

(Tanaka et al., 2005) or repeat forced swim stress (Ban-

erjee et al., 2005, 2010). Furthermore, RLN3 injected

i.c.v. or into hypothalamic nuclei of satiated rats pro-

duced an increase in feeding during the first hour post-

injection, and local injection into the paraventricular

hypothalamic nucleus (PVN) twice a day for 7 days or

i.c.v. infusion for 14 days resulted in increased food

intake and a sustained body weight gain (McGowan et al.,

2005, 2006, 2007; Hida et al., 2006).

In contrast to these comprehensive anatomical and ini-

tial functional studies in the rat, only limited reports on

the murine RLN3/RXFP3 system have appeared. And yet

the mouse represents an important experimental spe-

cies, due to the ability of genetically modified strains to

provide functional insights. Earlier studies identified

expression of RLN3 in a region of mouse brain homolo-

gous to the NI (Bathgate et al., 2002), and provided a

broad overview of RXFP3 mRNA and binding site distri-

butions (Liu et al., 2003b, 2005; Boels et al., 2004;

Sutton et al., 2006), but in the present study we aimed

to expand knowledge of the central RLN3/RXFP3 net-

works in the mouse. We mapped the distribution of

RLN3-positive neurons through the rostral-caudal extent

of the NI and described RLN3-positive neurons in

regions outside the NI, and produced a comprehensive

map of RLN3-positive nerve fibers. We mapped the dis-

tribution of RXFP3 throughout the forebrain axis and

conducted a semiquantitative assessment of the relative

regional abundance of RLN3-like immunoreactivity (-LI),

RXFP3 mRNA, and RXFP3 binding sites throughout the

entire brain.

Adult male C57BL/6J and mixed background

129S5:B6 wildtype (WT) and RLN3 knock-out (KO)/LacZ

Smith et al.

4018 The Journal of Comparative Neurology |Research in Systems Neuroscience

knock-in (KI) mice were used to map: 1) the distribution

of RLN3 gene expression by histochemistry for native

mRNA and LacZ reporter-gene activity; 2) RLN3-LI, using

an affinity-purified polyclonal antiserum (AS-R385-101)

against a conserved region of the RLN3 C-peptide (Ma

et al., 2007); 3) RXFP3 mRNA, using multiple [35S]-oligo-

nucleotide probes; and 5) RXFP3 binding sites, using

[125I]-R3/I5 (Sutton et al., 2004; Liu et al., 2005). The dis-

tribution of RXFP3 mRNA was examined at the cellular

level using nuclear emulsion, and these results were com-

pared to data in the open-access Allen Brain Institute

Gene Expression Atlas obtained using nonradioactive

RNA probes (Lein et al., 2007). Additionally, the RXFP3

protein distribution revealed by [125I]-R3/I5 binding was

analyzed in WT and RLN3 KO/LacZ KI mice. Preliminary

accounts of some of these data have appeared in

abstract form (Smith et al., 2006a,b) and in a recent con-

ference proceedings (Smith et al., 2009b).

MATERIALS AND METHODS

AnimalsAll procedures described were undertaken with the ap-

proval of the Howard Florey Institute Animal Welfare

Committee and in strict compliance with the ethical

guidelines issued by the National Health and Medical

Research Council of Australia. All efforts were made to

minimize the number of mice used and their discomfort.

Adult C57BL/6J male mice for the immunostaining and in

situ hybridization studies (18–25 g) were obtained from

the Australian Research Centre (Canning Vale, WA, Aus-

tralia). Mixed background (C57BL/6J � 129SV) RLN3

KO/LacZ KI mice, which express the LacZ reporter-gene

downstream, in-frame, and under the control of the en-

dogenous RLN3 promoter, were generated by Lexicon

Genetics (The Woodlands, TX). Adult (18–25 g) RLN3

KO/LacZ KI mice and WT littermates were generated via

heterozygous pairings and housed in the Howard Florey

Institute Animal Facilities. Mice were maintained on a

12:12-hour light dark cycle, with 3–4 mice per cage and

ad libitum access to standard chow and water.

Mouse genotypingWT (RLN3 (þ/þ)), heterozygous (RLN3 (þ/�)), and

KO (RLN3 (�/�)) mice were identified by polymerase

chain reaction (PCR) analysis of DNA extracted from tail

samples taken from each mouse using a REDExtract-N-

Amp Tissue PCR Kit (Sigma-Aldrich, Castle Hill, NSW, Aus-

tralia) following the manufacturer’s protocols. In the

RLN3 KO/LacZ KI mice used in this study, ablation of

functional RLN3 was achieved by deletion of exon 2 and

replacement with a LacZ/Neo cassette using targeting

vectors. For PCR identification of genotypes, a ‘‘forward’’

primer (which binds to an endogenous region upstream of

the LacZ/Neo cassette insertion site) was combined with

two downstream ‘‘reverse’’ primers, which bind to a

region within the endogenous WT genome that is deleted

in the KO allele (Reverse-WT primer), or to a region within

the LacZ/Neo cassette (Reverse-KO primer). The pres-

ence of a WT or KO allele resulted in the amplification of

a 621-bp or 281-bp product, respectively, which were

separated via gel electrophoresis. The sequences of pri-

mers used were as follows: Forward: 50-GGTTGGCAAGTAGTGTATGC-30; Reverse-WT: 50-TCTGATTTAGGGAGCCTAGC-30; Reverse-KO: 50-GCAGCGCATCGCCTTCTATC-30.The PCR reaction was conducted following the manufac-

turer’s protocols, using 50 pmol of forward primer and 25

pmol of each reverse primer, in a 20-lL reaction volume.

Amplification was conducted under the following condi-

tions: 94�C for 5 minutes; 35 cycles of 94�C for 40 sec-

onds, 58�C for 40 seconds, and 72�C for 2 minutes; fol-

lowed by a final 72�C for 10 minutes, and 4�C indefinitely.

Tissue preparationFor immuno- and X-GAL-histochemistry, mice were

killed by isoflurane inhalation overdose (IsoFLO; Abbott

Laboratories, Melbourne, VIC, Australia) and perfused

transcardially with 10 mL ice-cold phosphate buffer (PB)

solution (2.7 mM KCl, 11.2 mM Na2HPO4, 1.8 mM

KH2PO4, pH 7.4) followed by 50 mL ice-cold 4% parafor-

maldehyde in PB (pH 7.4) and then decapitated. Brains

were removed from the skull and immersed in fixative for

1 hour at 4�C before cryoprotection in 20% sucrose in PB

overnight at 4�C, coated in OCT embedding medium (Tis-

sue-Tek, Torrance, CA), and stored at �80�C. For in situ

hybridization and [125I]-R3/I5 binding assays, mice were

killed by isoflurane overdose and then decapitated.

Brains were quickly removed and embedded in OCT (Tis-

sue-Tek), frozen over dry ice, and stored at �80�C.

Antibody productionThe RLN3 antiserum used for mapping RLN3-LI was

raised against residues 85–101 of the C-chain of the

RLN3 propeptide that is identical in mouse and human,

with one conservative amino acid change from the rat

sequence (Bathgate et al., 2002; Burazin et al., 2002); it

has been characterized by our laboratory in studies of rat

and nonhuman primate brain (Ma et al., 2007, 2009b).

Thus, although this region of the RLN3 propeptide is pre-

dicted to be proteolytically cleaved during the processing

of mature RLN3, recent anatomical data suggest that the

distribution of RLN3-LI observed with this AS-R385-101C-chain antiserum is representative of the distribution of

mature RLN3 peptide (see Results) (Tanaka et al., 2005;

Ma et al., 2007). The specificity of the staining achieved

using affinity-purified AS-R385-101 has been demonstrated



in rats, where either omission of the primary antibody, use

of the preimmune serum, or preabsorption of the primary

antibody with 1 lg/mL pro-RLN3 C-peptide completely

abolished specific immunostaining of fibers/terminals (Ma

et al., 2007). In this study the specificity of this AS-R385-101antiserum was further validated by incubation with sections

through the medial septum of WT and RLN3 KO/LacZ KI

mice. These studies consistently resulted in strong immuno-

staining in the WT midline septal region and a total absence

of specific staining in equivalent sections from the RLN3 KO

mouse brain (Supporting Information Fig. S1).

ImmunohistochemistryCoronal sections (40 lm) through the rostrocaudal

brain axis of six brains were cut on a cryostat at �18�C(Cryocut 1800, Leica Microsystems, Heerbrugg, Switzer-

land) and collected in PB, pH 7.4. Free-floating sections

were preincubated in 70% methanol containing 0.6%

H2O2 for 30 minutes with gentle shaking, rinsed 3 � 10

minutes in PB, and then incubated in blocking buffer (10%

(v/v) normal horse serum and 0.3% (v/v) Triton X-100 in

PB) for 1 hour with shaking at room temperature. Sec-

tions were then incubated in PB containing polyclonal AS-

R385-101 RLN3 antiserum (dilution 1:2,500), 2% NHS,

0.3% Triton X-100, and 0.1% NaN3 (pH 7.4) with gentle

agitation for 72 hours at 4�C. Sections were washed 3 �10 minutes in PB and then incubated in PB containing bio-

tinylated antirabbit IgG (dilution 1:500, Vector Laborato-

ries, Burlingame, CA) with constant agitation for 1 hour at

room temperature, followed by 3 � 10 minute washes.

Sections were incubated in a 1:100 ABC/horseradish

peroxidase complex solution (Vectastain Elite, Vector

Laboratories) in PB for 1 hour at room temperature, fol-

lowed by 3 � 10 minute washes. Immunostaining was

visualized using a peroxidase-chromogen reaction by

placing the sections in 0.5 mg/mL 3, 30-diaminobenzidine

(DAB, Sigma-Aldrich) in PB containing 0.06% H2O2 for 1

minute before being washed for 3 � 10 minutes and

mounted onto gelatin-chrom alum-coated glass micro-

scope slides and dried overnight. Sections were then

dehydrated and cleared in ethanol and xylene and cover-

slipped using DePeX mounting media (Sigma-Aldrich).

X-GAL detection of LacZ reporter geneexpression in RLN3 KO/LacZ KI mice

Free-floating coronal sections (40 lm) from nine RLN3

KO/LacZ KI brains were incubated overnight at 37�C in

0.1% 4-chloro-5-bromo-3-indolyl-b-D-galactopyranoside(X-GAL, SciMAR, Templestowe, VIC, Australia), 2 mM

MgCl2, 5 mM EGTA, 0.01% (w/v) sodium deoxycholate,

0.02% (w/v) Nonidet P-40, 5 mM K3Fe(CN)6, and 5 mM

K4Fe(CN)6.6H2O in 0.1 M PB, pH 7.4. In brain regions

where the LacZ reporter gene was expressed under the

control of the endogenous RLN3 promoter, colorless solu-

ble X-GAL substrate was converted into a blue precipitate

that was visible in the neuronal soma and proximal proc-

esses. Sections were rinsed for 3 � 10 minutes in PB, pH

7.4, and mounted on 0.5% gelatin-chrom alum-coated

glass microscope slides, left to dry overnight, and then

counterstained in 0.01% neutral red solution for 2

minutes. After drying overnight, slides were dehydrated in

an ethanol series, cleared in xylene, and coverslipped

with DePeX mounting media (Sigma-Aldrich).

In situ hybridization histochemistryIn the current study, RLN3 mRNA was detected using

three DNA oligonucleotides complementary to nonoverlap-

ping regions of the mouse RLN3 sequence (50-GCACATCCGAATGAATCCGTCCATCCACTCCTCCGAGAC-30; 50-CAAGCAGAGCTGGCTCCTCCTGGCTCAAAGCCAATCTTC-30; 50-GTTGTAGCTCTGGGAGCGAGGCCTGAGCCTCAGACAGTA-30;NCBI Accession No. NM_173184). For detection of

RXFP3 mRNA, nine oligonucleotides complementary to

nucleotides of mouse RXFP3 cDNA were used (50-CAGTGAGTTGCCAGCGTTTGATGAGTTACATTGGCACCC-30; 50-GAAAACAGATCGTGGACATCTGTGTCTCGCTCTGACTGC-30;50-AGCAGGTGCCTGAGCTTTCTATGTAGACGCTCAAAAGGG-30; 50-GGCACAAACCACCCAGTAAACCGCGCTGATGAGG ATCCG-30; 50-GATGATGCTCAGCGGCAGCAGGAAGCCCAGCAGCACCTT-30; 50-CCAGGCGCTGCTGCTGCTCCTACTGCATCTGTTGTCCCA-30; 50-GCAGTAGAGGATCGG GTTGA

GGCAGCTGTTGGAGTGCGC-30; 50-CTTGGTGGTGGCGGTGAAAGGGCGCATGTTGGTGAGCGA-30; 50-AGCGCAGCCGAAGCCCAGATCAACCCACACAGCACCTTG-30; Accession

No. NM_178717). Although single oligonucleotide probes

are routinely used to detect abundant transcripts, exten-

sive experience within our laboratory (e.g., Ryan and

Gundlach, 1995), and other reports (e.g., Trembleau and

Bloom, 1995; Broide et al., 2004) demonstrate that multi-

ple oligonucleotide probes can be used to effectively

amplify the hybridization signal, with comparable or

improved specificity. Indeed, amplification is often essen-

tial for the successful detection of rare receptor tran-

scripts (e.g., Burazin et al., 2000).

Probes were produced commercially (Sigma-Aldrich)

and 30-end-labeled with [35S]-dATP (Amersham Life Sci-

ences, Amersham, UK) to a specific activity of �1–2 �109 dpm/lg, as described previously (Burazin et al.,

2002; Wisden and Morris, 2002). Using the GenBank

database, the sequence of each oligonucleotide was

checked for 100% homology to the target gene, less than

70% homology with other mammalian genes, and low sec-

ondary structure. Labeled probes were diluted in hybridiza-

tion buffer (1–5 pg/lL per probe) consisting of 50% (v/v)

formamide, 10% (v/v) dextran sulfate in 4 � SSC (1 �

Smith et al.


SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0). Dithio-

threitol (200 mM) was added to the solution to minimize di-

sulfide bridge formation and ‘‘nonspecific hybridization.’’

Brain sections from four mice were dehydrated in etha-

nol (70–100%) and delipidated in chloroform for 10

minutes to help decrease nonspecific ‘‘myelin binding’’ of

oligonucleotides during hybridization, and rinsed in 100%

ethanol (Burazin et al., 2002). Sections were then incu-

bated for 16 hours in hybridization buffer containing la-

beled 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 hybridiza-

tion was successfully displaced by this procedure (data

not shown). Labeled brain sections were coated with nu-

clear emulsion (Ilford K5, diluted 1:1 with dH2O; Ilford

Imaging, Melbourne, VIC, Australia) and exposed for 48

hours (RLN3 mRNA) or 20 weeks (RXFP3 mRNA) prior to

development, counterstaining with 0.01% thionin, and

analysis using brightfield and darkfield microscopy. Some

sections were exposed to Kodak BioMax film (Integrated

Sciences, Sydney, NSW, Australia) for 5 weeks for detec-

tion of RXFP3 mRNA.

Radioligand binding autoradiography[125I]-R3/I5 binding to mouse brain sections was

assessed as described (Sutton et al., 2004, 2006; Ma

et al., 2007). Coronal sections (14 lm) from three WT

and three RLN3 KO mice on gelatin-chrom alum-coated

glass microscope slides were preincubated for 15

minutes at room temperature in incubation buffer (20

mM HEPES, pH 7.4, 120 mM NaCl2, 0.22 mM KH2PO4,

1.3 mM CaCl2, 0.8 mM MgSO4). Sections were dried and

incubated for 60 minutes with 7 pM [125I]-R3/I5 (specific

activity, 2,200 Ci/mmol) in incubation buffer containing

0.5% bovine serum albumin and a protease inhibitor cock-

tail (Sigma-Aldrich). Nonspecific binding of [125I]-R3/I5

was determined in the presence of 1 lM unlabeled RLN3.

Following incubation, slides were washed in ice-cold incu-

bation buffer for 3 � 10 minutes and rinsed in ice-cold

deionized water. Sections were allowed to air-dry over-

night and were apposed to Kodak Biomax film (Integrated

Sciences) for 2 weeks prior to development and fixation

in an automated processor.

Analysis of relative density of RLN3-LI,RXFP3 mRNA, and [125I]-R3/I5 binding sites

The relative density of RLN3-LI in different areas was

scored by visual inspection of sections from six brains

according to a 6-point scale: (�) no RLN3-LI detected;

(þ/�) infrequent but detectable RLN3-LI; (þ) low density

of scattered RLN3-LI in the region; (þþ) moderate;

(þþþ) high; and (þþþþ) very high density of staining in

a distinct nucleus/region. For example, RLN3-LI density

was very high in the medial septum, high in the cingulate

cortex, moderate in the periaqueductal gray, and low in

the claustrum (see Results).

The distribution of neurons expressing RXFP3 was

mapped using data from three independent in situ hybrid-

ization studies: 1) radiolabeled riboprobe studies, where

signal intensity was detected using x-ray film (preliminary

data from these studies has been previously reported;

Sutton et al., 2006); 2) radiolabeled oligonucleotides

studies, where the regional density of positive neurons

and intensity of silver grain accumulation over individual

neurons was detected using nuclear photographic emul-

sion; and 3) nonradioactive riboprobe studies, where data

are available in the Allen Brain Atlas (Lein et al., 2007)

(www.brainmap.org). The distribution of RXFP3 mRNA

was first mapped separately using data from each of

these three studies, which revealed overlapping distribu-

tion patterns and few notable discrepancies. Therefore,

for the majority of coronal brain levels data from these

three separate studies were available, which was aver-

aged to produce the data presented.

Hybridization density within an area was rated inde-

pendent of neuronal size, i.e., a region with a moderate

number of large RXFP3-positive cells scored a similar

density value to a region with smaller yet more numerous

RXFP3-positive cells and a region with a high number of

large cells each with relatively reduced signal intensity.

The relative density of [125I]-R3/I5 binding sites was

scored by visual analysis of sections from six brains col-

lected in the present study and images from an earlier

study (Sutton et al., 2006). RXFP3 mRNA and [125I]-R3/I5

binding site densities were scored using a 6-point scale:

(�) no RXFP3 mRNA or binding sites detected; (þ/�)

scarce, (þ) low, (þþ) moderate, (þþþ) high or (þþþþ)

very high density of RXFP3 mRNA or binding sites in a dis-

tinct nucleus/region. For example, the RXFP3 mRNA and

binding site density was very high in the medial nucleus

of the amygdala, high in the bed nucleus of the stria ter-

minalis, moderate in the nucleus incertus, and low in the

auditory cortex (see Results).

Photography and image productionBrightfield and darkfield digital images of RLN3 immu-

nostaining and RXFP3 mRNA-associated nuclear emul-

sion autoradiograms were captured using MCID-M2 soft-

ware (Imaging Research, St Catharine’s, ON, Canada) on

a Nikon Microphot SA microscope (FSE, Melbourne, VIC,

Australia) equipped with a Sony CCD camera. X-ray film

images were viewed using a Sony XC-77 camera mounted

above a light-box (Northern Lights; Berthold Australia,

Bundoora, VIC, Australia). All illustrative images were



archived as high-resolution images (at least 500 dpi) and

were cropped, adjusted for contrast, had obvious arti-

facts removed, and were arranged and labeled using

Adobe Photoshop 7.0 and Adobe Illustrator 10 for Win-

dows (Adobe Systems, San Jose, CA). Schematics were

created using Allen Brain Atlas software (Dong, 2008) to

illustrate the RLN3 neuron distribution in the NI and the

Mouse Brain Atlas software (Franklin and Paxinos, 1997)

to illustrate the distribution of RLN3-LI, RXFP3 mRNA and

[125I]-R3/I5 binding sites, using Adobe Photoshop 7 and

Illustrator 10.

RESULTS

The present study identified an enrichment of RLN3

mRNA and RLN3-LI in the NI of mouse brain (Fig. 1), simi-

lar to observations in the rat (Burazin et al., 2002; Tanaka

et al., 2005; Ma et al., 2007). High levels of RLN3 gene

expression were reflected by the detection of high levels

of mRNA by in situ hybridization of [35S]-oligonucleotides

and high levels of X-GAL staining, reflecting activity of the

LacZ reporter gene in RLN3 KO/LacZ KI mice (Fig. 2).

RLN3-LI was not detectable in RLN3 KO mice (Supporting

Information Fig. S1), but was present in neuronal cell

bodies within the NI in WT mice (Fig. 3). RLN3 was also

expressed by a number of small populations of neurons in

the periaqueductal gray, in a midbrain area dorsal to the

substantia nigra, and in the pontine raphe nucleus (Fig. 4).

RLN3-LI was observed in extensive networks of axons and

nerve terminals throughout the forebrain (Figs. 5–9) in a

pattern that overlapped the distribution of RXFP3 mRNA

(Figs. 5, 10, 11) and [125I]-R3/I5 binding sites (Figs. 5, 12);

again paralleling data from the rat (Sutton et al., 2004;

Tanaka et al., 2005; Ma et al., 2007). Further detailed

descriptions and semiquantitative assessments of these

findings are provided below (see Fig. 5; Table 1).

Distribution of RLN3 neurons in C57BL/6Jand RLN3 KO/LacZ KI mouse brain

RLN3-positive neurons were identified using three

methods. First, in situ hybridization was used to directly

detect RLN3 mRNA. The short 2-day exposure time to nu-

clear emulsion required to achieve a photographic image

indicates that RLN3 mRNA is highly abundant. Second,

RLN3 expression was detected indirectly via LacZ re-

porter gene activity, as a blue X-GAL associated precipi-

tate in the soma and proximal processes of NI neurons in

RLN3 KO/LacZ KI brain sections. Notably, X-GAL staining

offers advantages over in situ hybridization, such as

increased morphological detail and the ability to work

without any potential radioactive hazard. The observed

overlap in the distribution of RLN3 mRNA and reporter

gene activity suggests that X-GAL staining faithfully iden-

tifies neurons that normally express RLN3 in WT mice.

Lastly, immunohistochemistry using a polyclonal RLN3

antiserum detected RLN3-LI in cytoplasm and proximal

processes of NI neurons. The RLN3-LI present in NI soma

was of sufficient abundance to be detected without pre-

treatment of the mice with colchicine to enhance the cel-

lular accumulation of peptide (Ma et al., 2007). All three

techniques revealed overlapping distributions and pro-

vide an accurate description of the distribution of RLN3-

positive neurons. This distribution of RLN3-positive neu-

rons was summarized on Allen Brain Atlas schematic

images (Dong, 2008), as this atlas provides several plates

within the restricted rostral-caudal extent of the NI (Fig. 1).

Therefore, in these plates the borders of the NI illustrated

are those provided in the Allen Brain Atlas, but we have

noted a possible lack of consensus on whether the most

midline of cells within these borders are part of the NI or

part of the central gray. For example, Franklin and Paxi-

nos (1997) do not include cells along the midline in the

nucleus O (or NI) and several reports in the rat are con-

sistent with the bilateral nature of the NI (Goto et al.,

2001; Olucha-Bordonau et al., 2003; Tanaka et al., 2005;

Ma et al., 2007). Furthermore, several other genes

enriched in the mouse NI also display a bilateral appear-

ance (see, e.g., CRF binding protein and E430002G05Rik;

www.brainmap.org; Lein et al., 2007).

RLN3-positive neurons in the NIThe majority of RLN3-positive neurons were observed

within the NI, located medioventral to the locus coeruleus

and along the medial and ventral border of the dorsal teg-

mental nucleus (Fig. 1). At the rostral border of the mouse

NI defined in the Allen Brain Atlas (bregma �5.25 mm), a

high density of RLN3-positive neurons was present bilat-

erally in the ventral part of the nucleus, near the midline

and just dorsal to the medial longitudinal fasciculus, while

RLN3 neurons in more lateral and dorsal regions were

less tightly packed (Figs. 1A, 2). Neurons along the adja-

cent midline region were RLN3-negative. More caudally, a

high density of RLN3-positive cells was observed through-

out the extent of the NI (bregma �5.38 mm; Figs. 1B, 3),

with negative neurons present along the midline. Addi-

tionally, a low density of positive cells was observed at

the border of the NI and the pontine central gray (Dong,

2008).

The distribution of RLN3-positive neurons further cau-

dally (bregma �5.45 mm; Fig. 1C) was reminiscent of the

characteristic topography described in the rat, where the

NI consists of a tightly clustered and medial pars com-

pacta and a more lateral and diffuse pars dissipata

(Tanaka et al., 2005; Ma et al., 2007). At the caudal

boundary of the NI (bregma �5.55 mm), two diffuse pop-

ulations of RLN3-positive neurons were observed—a

Smith et al.


midline population that extended dorsally to include

some periventricular cells and a lateral group that

spanned the lateral border of the NI and continued dor-

sally into the pontine central gray (Fig. 1D). Interestingly,

low densities of RLN3-positive neurons were also

observed in periventricular regions caudal to the defined

Figure 1. Schematic illustration of the distribution and relative density of neurons containing RLN3 in coronal sections through the rostrocaudal

extent of the mouse NI and surrounding regions, based on the similar distributions observed using in situ hybridization, LacZ staining, and immuno-

histochemical studies. Neurons positive for RLN3 mRNA, LacZ staining, and RLN3-LI are indicated by red circles and regions that contained high

and low densities of positive neurons are designated by dark and light shading, respectively. See Results for further details. The distribution of RLN3-

positive cells was overlaid onto schematic images taken from the Allen Institute Brain Atlas, plates 106–111 (Dong, 2008), corresponding to bregma

(A)�5.25 mm, (B) �5.38 mm, (C) �5.45 mm, (D) �5.55 mm, (E)�5.65 mm, (F) �5.78 mm. For abbreviations, see list. Scale bar¼ 200 lm.



NI in the Allen Atlas (bregma �5.65 and �5.78 mm; Fig.

1E,F) (Dong, 2008).

RLN3-positive neurons outside the NISeveral small populations of RLN3-positive neurons

were detected in the anterior periaqueductal gray, in the

pontine raphe nucleus, and in a region dorsal to the lat-

eral substantia nigra, and coinciding with or adjacent to

the lateral terminal and peripeduncular nuclei (Fig. 4A–

C). This distribution was observed with X-GAL staining of

RLN3 KO/LacZ KI brain sections (Fig. 4D–F), as RLN-LI

present in sections from C57BL/6J mice (Fig. 4G–I), and

was also observed in adult C57BL/6J mouse brain sec-

tions hybridized with a digoxigenin-labeled riboprobe

targeting RLN3 mRNA and detected using alkaline phos-

phatase (Fig. 4J–L; see Allen Brain Atlas; www.brainma-

p.org) (Gundlach et al., 2009). The authenticity of these

highly correlated findings is further supported by similar

observations in the rat (Tanaka et al., 2005; Ma et al.,

2007).

Figure 2. Expression of RLN3 in the NI detected by in situ hybridization of [35S]-labeled oligonucleotides in a C57BL/6J mouse brain and

X-GAL staining of LacZ reporter-gene expression in RLN3 KO/LacZ KI mouse. A,B: Low-magnification brightfield and darkfield images of a

representative thionin-counterstained coronal brain section through the rostral extent of the NI (bregma �5.25 mm), illustrating the pres-

ence of RLN3 mRNA in dispersed neurons in the NI (see Fig. 1A). C: High-magnification brightfield image illustrating clusters of silver

grains over individual neurons in the NI. The asterisk in B,C marks the same position on the section. D: Low-magnification brightfield

image of a representative nuclear fast red counterstained coronal brain section through the same rostral extent of the NI as above (bregma

�5.25 mm), taken from the brain of an RLN3 KO/LacZ KI mouse. The presence of blue precipitate indicative of RLN3-associated LacZ

reporter-gene expression is visible within a cluster of NI neurons, as above. E: Higher-magnification image of the same section more clearly

illustrating the characteristic bilateral distribution of neurons that contain RLN3-associated staining within the NI at this level. F: High-

magnification image illustrating the morphology of individual RLN3-expressing neurons in the NI and the presence of strong blue X-GAL stain-

ing in their soma and proximal extensions. For abbreviations, see list. Scale bars ¼ 200 lm in A,B,D; 100 lm in E; 25 lm in C; 30 lm in F.

[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Smith et al.


Specificity of RLN3 antiserum andverification of RLN3 deficiencyin KO mice

Brain sections from RLN3 KO/LacZ KI mice did not dis-

play any RLN3-LI in regions such as the medial septum

following incubation with the characterized polyclonal

RLN3 antiserum, despite high levels observed in equiva-

lent sections from WT mice (Supporting Information

Fig. S1). This finding strongly supports the specificity of

the antiserum for RLN3 (Saper and Sawchenko, 2003),

and is in line with our earlier reports in the rat and maca-

que brain (Ma et al., 2007, 2009b). The data also confirm

that RLN3 is indeed deficient in this strain of mice.

Distribution of RLN3-LI in neuronalprojections of mouse brain

Punctate RLN3-LI staining characteristic of nerve fibers

and terminal boutons was broadly distributed throughout

the mouse brain, in line with the trafficking, targeting, and

storage of RLN3 observed in the rat (Tanaka et al., 2005).

The distribution of RLN3-LI observed in six brains was col-

lated and plotted onto schematic brain sections (Franklin

and Paxinos, 1997; Fig. 5), and the relative density of

RLN3-LI in different brain regions was semiquantitatively

scored (Table 1). Descriptions of the topography of RLN3-

LI throughout the neuraxis follow, with reference to sum-

mary data and relevant micrographs (Figs. 6–9).

Figure 3. RLN3-LI in the NI of a C57BL/6J mouse brain labeled by a polyclonal C-peptide antiserum (AS-R385-101) and visualized by DAB

immunohistochemistry. A,B: Low- and high-magnification brightfield images of a representative coronal brain section through the ‘‘central’’

region of the NI (bregma �5.38 mm), illustrating the presence of RLN3-LI in dispersed neurons and their proximal processes in the NI

(see Fig. 1B). C,D: Darkfield images of the same section, illustrating the distribution of nerve fibers/terminals that contain RLN3-LI within

the NI region in addition to the nearby dorsal tegmental nucleus and area adjacent to the fourth ventricle. The region shown in B,D is out-

lined in A,C. For abbreviations, see list. Scale bars ¼ 100 lm in A,C; 50 lm in B,D. [Color figure can be viewed in the online issue, which

is available at www.interscience.wiley.com.]



Rhinencephalon and telencephalonLow densities of nerve axons/terminals containing

RLN3-LI were detected in the anterior olfactory nucleus,

ependyma, and surrounds of the olfactory ventricle (Fig.

5A). A high density was detected in the dorsal transition

zone, and a low to moderate density of fibers was

observed in the cortical areas medial to the forceps minor

of the corpus callosum, including the dorsal peduncular,

Figure 4. Distribution of neuronal cell bodies displaying LacZ expression, RLN3-LI and RLN3 mRNA in the anterior periaqueductal gray

(left column), a region dorsal to the substantia nigra (central column), and the pontine raphe nucleus (right column). A–C: Schematic rep-

resentation of locations of RLN3 neurons in (A) periaqueductal gray (bregma �2.54 mm), (B) a region dorsal to the substantia nigra

(bregma �3.80 mm), and (C) the pontine raphe nucleus (bregma �5.15 mm). Schematics adapted from Franklin and Paxinos (1997).

D–F: High-magnification brightfield images of strongly stained neurons positive for LacZ expression (arrows). G–I: High-magnification bright-

field images of darkly stained neurons positive for RLN3-LI (arrows). J–L: High-magnification brightfield images of darkly stained neurons

positive for RLN3 mRNA (arrows), adapted from images available from the Allen Brain Atlas (www.brainmap.org). For abbreviations, see

list. Scale bars ¼ 1.0 mm in A–C; 25 lm in D–F; 20 lm in G–I; 100 lm in J–L.

Smith et al.


Figure 5. Schematic illustration of the distribution and relative densities of RLN3-LI, RXFP3 mRNA, and RXFP3 binding sites in coronal

sections through the rostrocaudal extent of the C57BL/6J mouse brain. A–P: A series of coronal drawings, adapted from the stereotaxic

atlas of (Paxinos and Watson, 2007), illustrating the distribution and relative density of RLN3-LI (fine red lines), RXFP3 mRNA (blue dots),

and [125I]-R3/I5 binding sites (green areas, with light shading indicating a low density, and dark shading indicating a high density of bind-

ing sites). The position (in mm) of each plate relative to bregma is: A 2.68; B 1.98; C 1.18; D 0.62; E 0.02; F �0.94; G �1.34; H �1.94; I

�2.54; J �2.92; K �3.80; L �4.48; M �4.96; N �5.40; O �5.88; P �7.64. The distribution of RXFP3 mRNA was determined by combin-

ing data generated in the current study with data publicly available in the Allen Brain Atlas (www.brainmap.org). For abbreviations, see list.

Scale bar ¼ 1.0 mm.



infralimbic, prelimbic, and rostral cingulate cortices (Fig.

5B; Table 1).

The highest densities of RLN3 immunostained ele-

ments observed within the forebrain were present in the

septal regions (Fig. 5C–E). In particular, a very high den-

sity was detected in the medial septum (Fig. 6A,B), lamb-

doid septal zone, and also in the lateral septum, where

fibers were concentrated in the intermediate area, with

considerably less staining present in the dorsal and ven-

tral regions (Fig. 6D–F). This dense plexus of fibers

appeared to course dorsally from the horizontal and verti-

cal limbs of the nucleus of the diagonal band (Fig. 6A,C),

Figure 5. (Continued)

Smith et al.


through the medial septum, and continued to travel dor-

sally to innervate the septohippocampal (Fig. 6D) and

septofimbral nuclei (Fig. 6F) with a high density of fibers/

terminals.

A considerable cortical RLN3 innervation was also

observed, especially in midline structures (Fig. 5B–L).

Rostrally, a low and high density of fibers were observed

in the medial orbital cortex and dorsal tenia tecta, respec-

tively (Fig. 7A), while most prominently, a high density of

fibers were observed across the entire rostral-caudal

length of the cingulate and retrosplenial cortices (Fig.

7B,C,E) that appeared to represent the majority of the de-

tectable cortical innervation. A low density of fibers was

also detected at the medial border of the secondary

motor and visual cortices (Fig. 7C) and in the claustrum

(Fig. 7D) and dorsal endopiriform cortex, while more cau-

dally, a low density of long fibers was detected sweeping

into the lateral entorhinal cortex (Fig. 7F).

A low density of fibers was observed in the bed nucleus

of the stria terminalis (Figs. 5E, 8A), while in the amyg-

dala, various nuclei also displayed low densities of RLN3-

LI (Fig. 5F–J), such as the amygdalostriatal transition

area, and the central, medial, and basomedial nuclei (Fig.

8B,C). In contrast, the adjacent substantia innominata

displayed a high density of fibers (Figs. 5G–H, 8C).

RLN3-LI in the hippocampus was abundant in both the

dorsal and ventral regions, and generally appeared re-

stricted to distinct cell layers (Fig. 5F–K). At the anterior

extent of hippocampus, a moderate density of fibers was

detected in the dorsal fornix and surrounding alveus (Fig.

7B). Throughout the entire CA fields a high and moderate

density of fibers was detected within the oriens and bor-

dering alveus layer, respectively; and a low density was

detected in the pyramidal cell layer (Fig. 8D,E). The lim-

ited amounts of RLN3-LI detected in other regions of the

CA fields resulted in a low average density rating across

the entire CA1 and CA2 fields, while the CA3 field dis-

played a low to moderate density (Table 1). Within the

dentate gyrus, RLN3-LI was largely restricted to the poly-

morph layer (Fig. 8F) in a moderate overall density,

although staining was more obvious in posterior sections.

DiencephalonThe hypothalamus contained numerous distinct

regions with highly enriched RLN3-LI, while others were

apparently not strongly innervated (Fig. 5E–J). A low

Figure 6. Distribution of RLN3-LI in the septal region of the C57BL/6J mouse brain. Coronal sections were incubated with the polyclonal

RLN3 antiserum (AS-R385-101) and labeling was visualized by DAB immunohistochemistry and viewed under darkfield illumination. A: Low-

magnification image of a representative coronal brain section illustrating the distribution of nerve fibers/terminals that contain RLN3-LI

within the medial septum and diagonal band; B,C: Higher-magnification images of medial septum, vertical, and horizontal diagonal band;

D,E: Low- and high-magnification images of the distribution of RLN3-LI within the lateral septum, F: Low-magnification image of RLN3-LI

within the septofimbrial and dorsal lateral septal nuclei. For abbreviations, see list. Scale bars ¼ 700 lm in A; 250 lm in B,C,D,F; 100

lm in E. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



density of RLN3-LI was observed in prominent hypothala-

mic regions including the paraventricular hypothalamic

nucleus, the ventromedial hypothalamus (Fig. 9A), and

the tuberomammillary nucleus. In contrast, a very high

density of fibers were observed within the posterior hypo-

thalamic area (Fig. 9B), and lateral hypothalamus/medial

forebrain bundle (Fig. 9C) where the majority of fibers

coursed in a caudal-rostral direction. Additionally, high

densities of fibers were detected in the supraoptic nu-

cleus and bordering regions, and in the supramammillary

nucleus (Fig. 9D), while a moderate density was observed

in the lateral mammillary nucleus, and a low number of

fibers were evident in the medial and premammillary

nuclei. At the rostral extent of the hypothalamus, a

moderate density of fibers was observed in the lateral

preoptic area, with only scarce fibers in the medial pre-

optic area.

The thalamus also displayed prominent RLN3-LI that

was most apparent in lateral regions (Fig. 5F–K). Very

high densities were observed in both the dorsal and lat-

eral terminal nuclei of the accessory optic tract (Fig.

9E,F), and fibers from the latter projected dorsally around

the basal cerebral peduncle resulting in a very high and

high density of fibers in the peripeduncular nucleus and

posterior intralaminar thalamic nucleus, respectively (Fig.

9G). Furthermore, RLN3-LI was apparent in various

regions within the lateral geniculate complex (Fig. 5H,I),

in high densities in the ventrolateral nucleus (Fig. 9H);

moderate densities in the intergeniculate, subgeniculate,

and suprageniculate areas; and low densities in the

medial and dorsolateral areas (Fig. 9E). Moderate den-

sities were also observed within the zona incerta. In con-

trast, low densities of RLN3-LI were detected in more

medial regions of the thalamus, such as the habenula,

and the paraventricular, reuniens, rhomboid, and precom-

missural nuclei.

MesencephalonThe majority of RLN3-LI observed within the midbrain

was associated with either the periaqueductal gray or

superior colliculus (Fig. 5I–M). In the anterior periaque-

ductal gray (Fig. 5I,J), in addition to cell bodies containing

RLN3-LI (Fig. 4), a moderate density of fibers coursed

mainly in the dorsal-ventral plane (Fig. 9J) that extended

further dorsally into the posterior commissure, posterior

pretectal, and olivary pretectal nuclei. Throughout the

more caudal periaqueductal gray (Fig. 5K–M) a high den-

sity of RLN3-positive fibers radiated in an outward

Figure 7. Distribution of RLN3-LI in cortical regions of C57BL/6J mouse brain. Coronal sections were incubated with the polyclonal RLN3

antiserum (AS-R385-101) and labeling was visualized by DAB immunohistochemistry and viewed under darkfield illumination. A: High-magnifi-

cation image of a representative coronal brain section illustrating the distribution of nerve fibers/terminals that contain RLN3-LI within the

dorsal tenia tecta; B,C: High-magnification images of the distribution of RLN3-LI within the midline cingulate cortex and anterior hippocam-

pus; D: RLN3-LI within the claustrum; E: RLN3-LI in the retrosplenial cortex, F: RLN3-LI in the entorhinal cortex. For orientation, the mid-

line (dotted line, m/l) or lateral direction (arrow, lat) is indicated. For abbreviations, see list. Scale bar ¼ 250 lm. [Color figure can be

viewed in the online issue, which is available at www.interscience.wiley.com.]

Smith et al.


direction from the aqueduct into the dorsomedial and

dorsolateral regions, while the lateral and ventrolateral

regions displayed a moderate density of fibers (Fig. 9K).

In the superior colliculus, a high density of fibers was

observed in an ‘‘m"-shaped band, which spanned the

entire width of the structure and encompassed the inter-

mediate gray and white layers (Figs. 5K,L, 9L). The den-

sity of fibers was reduced dorsally and ventrally from this

central band with the adjacent optic nerve and deep gray

layers containing moderate densities, while the outer su-

perficial gray and deep white layers displayed only low

densities of immunoreactive elements (Table 1).

Other smaller, yet notable, midbrain areas were also

rich in RLN3-LI. A high density of RLN3-LI was detected in

the interpeduncular nucleus, where it was concentrated

in the outermost lateral, dorsolateral, and dorsomedial

subnuclei (Figs. 5K, 9I; Table 1), while a moderate density

of fibers was observed in the ventral tegmental area (Fig.

5J,K). Finally, the rostral linear raphe displayed only a low

density of RLN3-LI, while both the caudal linear and dor-

sal raphe nuclei received very high densities of fibers,

with the latter appearing to originate posteriorly before

being funneled between the medial longitudinal fasciculi

(Fig. 5M).

RhombencephalonAs expected, very high densities of RLN3-LI were

detected within the NI (see Fig. 3) from where fibers were

observed to project to various other proximal hindbrain

targets. The majority of fibers projecting rostrally inner-

vated the raphe system (Fig. 5L–N), resulting in very high

densities of RLN3-LI in the median and paramedian raphe

nuclei, and high densities in the pontine raphe nucleus

and raphe cap. In contrast, the tegmental system was

largely encircled by RLN3-LI (Fig. 5M,N), resulting in gener-

ally low RLN3-LI among its subnuclei (Table 1). Several

exceptions to this were observed, however: the dorsal and

anterior tegmental nuclei displayed moderate and high

densities of RLN3-LI (see Fig. 3), which may be due to their

close proximity to the RLN3 rich NI and median raphe,

respectively; and the pedunculopontine tegmental nucleus

received a moderate density of RLN3-LI (Fig. 5M).

A high density of fibers was observed projecting later-

ally from the NI to the neighboring central gray; although

interestingly, RLN3-LI was absent from the locus coeru-

leus (Fig. 5N). Fibers were observed to extend dorsally

from the NI to the edge of the fourth ventricle, before ei-

ther diverging laterally to innervate the lateral parabra-

chial nucleus with a low density of RLN3-LI (Fig. 5N), or

Figure 8. Distribution of RLN3-LI in the amygdala and hippocampal region of the C57BL/6J mouse brain. Coronal sections were incubated

with the polyclonal RLN3 antiserum (AS-R385-101) and labeling was visualized by DAB immunohistochemistry and viewed under darkfield

illumination. High-magnification images of representative coronal brain sections illustrating the distribution of nerve fibers/terminals con-

taining RLN3-LI within A: bed nucleus of the stria terminalis; B: amygdala; C: substantia innominata; D,E: hippocampus oriens layer;

F: hilus of dentate gyrus. For orientation, the lateral direction (arrow, lat) is indicated. For abbreviations, see list. Scale bars ¼ 250 lm in

A–D,F; 100 lm in E. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]



projecting further caudally where a moderate density of

fibers was observed in the parvicellular medial vestibular

and prepositus hypoglossal nuclei (Fig. 5O). Finally, low

densities of RLN3-LI were detected further caudally in the

nucleus of the solitary tract and medial inferior olive (Fig.

5P).

Figure 9. Distribution of RLN3-LI in the diencephalon and mesencephalon of the C57BL/6J mouse brain. Coronal sections were incubated with

the polyclonal RLN3 antiserum (AS-R385-101) and labeling was visualized by DAB immunohistochemistry and viewed under darkfield illumination.

High-magnification images of representative coronal brain sections illustrating the distribution of nerve fibers/terminals containing RLN3-LI

within A: ventromedial hypothalamus; B: posterior hypothalamus; C: lateral hypothalamus/medial forebrain bundle, D: supramammillary nucleus;

E: dorsal terminal nucleus of the accessory optic tract; F: lateral terminal nucleus of the accessory optic tract, G: peripeduncular nucleus; H:

ventrolateral geniculate nucleus; I: interpeduncular nucleus; J: anterior periaqueductal gray; K: posterior periaqueductal gray, L: superior collicu-

lus. For orientation, the midline (dotted line, m/l) or lateral direction (arrow, lat) is indicated. For abbreviations, see list. Scale bars ¼ 250 lmin A–D,F–L; 100 lm in E. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Smith et al.


No RLN3-LI was observed within the cerebellum and

the distribution of RLN3-LI in the spinal cord was not

examined.

Distribution of neurons expressingRXFP3 mRNA

The distribution of RXFP3 mRNA was comprehensively

mapped using data collected in three independent in situ

hybridization studies, which all yielded overlapping distri-

butions that were averaged (see Materials and Methods

for details). The regional distribution of RXFP3 mRNA was

plotted onto schematic brain sections (Fig. 5), and the rel-

ative regional densities of RXFP3 mRNA were semiquanti-

tatively scored (Table 1). A description of this distribution

is provided below, with reference to illustrative autoradio-

grams (Figs. 10, 11).

Rhinencephalon and telencephalonNumerous regions within the telencephalon contained

high densities of RXFP3 mRNA-positive cells, including

the hippocampus and associated areas (Figs. 5F–K, 10),

the medial septum (Figs. 5C–E, 11A), the bed nucleus of

the stria terminalis (Fig. 11B), and the ventral part of the

lateral septum; while the horizontal limb of the diagonal

band and other septal regions displayed moderate den-

sities (Table 1). In hippocampus, high densities of RXFP3

mRNA were present throughout the oriens and pyramidal

Figure 10. RXFP3 mRNA in neurons of the hippocampus and amygdalohippocampal area of the C57BL/6J mouse. Coronal brain sections

were incubated with specific [35S]-labeled oligonucleotides and hybridization was detected associated with individual neurons using nu-

clear photographic emulsion. A: Low-magnification darkfield image of the ventral hippocampus and the amygdalohippocampal area.

B: High-magnification brightfield image of the amygdalohippocampal area. C,D: High-magnification brightfield images of the pyramidal cell

layer, illustrating the presence of clustered silver grains over individual interneurons (arrows) counterstained with thionin. Boxes indicate

regions that are enlarged in subsequent plates. For orientation, the lateral direction (arrow, lat) is indicated. For abbreviations, see list.

Scale bars ¼ 500 lm in A; 120 lm in B; 80 lm in C; 40 lm in D.



cell layer, resulting in an overall moderate density of

RXFP3 mRNA expression throughout all the large CA

fields (Table 1). In the ventral hippocampus, cells that

expressed a very high level of RXFP3 mRNA displayed to-

pography similar to that of large inhibitory interneurons

(Fig. 10A,C,D). In the dentate gyrus, high densities of pos-

itive cells were detected in the polymorph layer and adja-

cent to or overlying the granule cell layer (Fig. 11C).

The amygdala also displayed strong RXFP3 mRNA

expression (Fig. 5G–K). High densities were detected in

the central nuclei, and along the medial border of the lat-

eral fork of the external capsule, corresponding to the lat-

eral nucleus. A large cluster of RXFP3 mRNA-positive cells

was observed just posterior and ventral to these neurons,

which resulted in an overall high density in the basomedial

and basolateral nuclei, while high and very high densities

were observed in the posteromedial cortical and medial

nuclei, respectively (Fig. 11D). The posterior amygdaloid

region was characterized by a ‘‘boomerang-shaped’’ group

of positive cells comprising the amygdalohippocampal and

amygdalopiriform transition areas, which displayed very

high and moderate densities, respectively (Figs. 5J, 10A,B).

A low density of RXFP3 mRNA expression was detected

throughout much of the mouse forebrain, including the

anterior olfactory nucleus and external plexiform layer of

the olfactory bulb, dorsal tenia tecta, and dorsal transi-

tion zone. Of particular note, throughout the entire rostro-

caudal extent of the cerebral cortex, a low density of

Figure 11. Distribution of RXFP3 mRNA in the forebrain of the C57BL/6J mouse detected by in situ hybridization histochemistry. Coronal

brain sections were incubated with specific [35S]-labeled oligonucleotides and labeling was detected using nuclear photographic emulsion

and visualized under darkfield illumination. Specific hybridization was widely distributed throughout the forebrain axis, including A: medial

septum; B: bed nucleus of the stria terminalis; C: hilus of dentate gyrus; D: amygdala; E,F: periventricular hypothalamic nucleus; G: para-

ventricular hypothalamic nucleus; H: interpeduncular nucleus; I: inferior colliculus. For orientation, the midline (dotted line, m/l) or lateral

direction (arrow, lat) is indicated. For abbreviations, see list. Scale bars ¼ 700 lm in B–D; 250 lm in A,E,G–I; 100 lm in F.

Smith et al.


RXFP3 mRNA was detected in the outer layers of almost

all regions, while the piriform cortex displayed a moderate

density (Fig. 5B–L; Table 1).

DiencephalonThe most prominent site of hypothalamic RXFP3 mRNA

expression was the paraventricular and supraoptic hypo-

thalamic nuclei, which displayed very high densities (Figs.

5F, 11G). High densities were also observed within the

periventricular hypothalamic nucleus (Fig. 11E,F), while

moderate densities were detected in the lateral hypo-

thalamic area / medial forebrain bundle, posterior hypo-

thalamus, and supramammillary nucleus (Fig. 5G–J).

Thalamic RXFP3 mRNA expression was generally low

(Fig. 5F–K), except for very high and high densities in the

habenula and subparafascicular nucleus, respectively,

and moderate densities in the precommissural nucleus

and ventrolateral geniculate nucleus. A cluster of RXFP3

mRNA-positive cells was also observed dorsal to the cere-

bral peduncle, resulting in very high and high densities in

the peripeduncular and posterior intralaminar nuclei,

respectively (Table 1).

MesencephalonRXFP3 mRNA expression was widespread throughout

the midbrain (Fig. 5I–M). The periaqueductal gray was

Figure 12. Distribution of RXFP3 binding sites in the forebrain of a C57BL/6J mouse. Representative low-resolution X-ray film autoradio-

grams of [125I]-R3/I5 binding sites in selected coronal sections of the forebrain at the level of A: bed nucleus stria terminalis; B: hypo-

thalamic paraventricular nucleus; C: anterior amygdala and dorsal hippocampus; D: posterior amygdala and anterior periaqueductal gray;

E: ventral hippocampus; F: superior colliculus. No specific binding was observed in near adjacent sections incubated with radioligand plus

100 nM unlabeled human RLN3 (data not shown). For abbreviations, see list. Scale bar ¼ 1.25 mm.



TABLE 1.

Regional Distribution and Relative Density of RLN3-LI, RXFP3 mRNA, and RXFP3 Binding Sites in Mouse Brain

Brain region RLN3-LI RXFP3 mRNA RXFP3 binding sites

RhinencephalonOlfactory bulb

anterior olfactory n. þ þ þependyma and olfactory ventricle þ � �external plexiform layer � þ �glomerular layer � � þinternal granule cell layer � � �

Telencephalonaccumbens n., shell þ/� þ �dorsal tenia tecta þþþ þ þdorsal transition zone þþþ � þsubstantia innominata þþþ þþ þventral pallidum � þ �ventral tenia tecta þ � þAmygdala

amygdalohippocampal area � þþþþ þþþþamygdalopiriform transition area � þþ þþþþamygdalostriatal transition area þ þ/� �anterior amygdaloid area � þ �anterior cortical amygdaloid n. � þ/� þbasolateral n. þ/� þþþ þþþbasomedial n. þ þþþ þþþþcentral n. þ þþþ þþcortex-amygdala transition area � þ/� þlateral n. þ/� þþþ þþþmedial n. þ þþþþ þþþþposterolat cortical amygdaloid n. � þ þposteromed cortical amygdaloid n. � þþþ þþ

Bed nucleus of the stria terminalis

intraamygdaloid division þ þþ þþþlateral division þ þþþ þþmedial division þ þþþ þþþventral division � þ þ/�

Cerebral cortex

auditory cortex � þ þcingulate cortex þþþ þ �claustrum þ þ/� �dorsal peduncular cortex þþ þ/� �ectorhinal cortex � þ þendopiriform n. dorsal þ þ þ/þþendopiriform n. ventral � þ þþþentorhinal cortex þ þ þfrontal association cortex � þ/� þ/�infralimbic cortex þ þ �insular cortex � þ þorbital cortex, medial þ/þþ þ þ/�perirhinal cortex � þ þpiriform cortex � þþ þ/þþposterior parietal associative area � þ þprelimbic cortex þ þ/� þ/�primary motor cortex þ/� þ þretrosplenial cortex þþþ þ �secondary motor cortex þ þ þsomatosensory cortex � þ þsubiculum þ þ þ/�temporal cortex � þ þvisual cortex þ þ þ

Hippocampal formation

alveus þþ � þ/�CA1 field þ þþ þþCA2 field þ þ þþCA3 field þ/þþ þþ þþDG, molecular layer þ/� � þDG, polymorph layer þþ þþþ þþþ

Smith et al.


TABLE 1 (Continued)


DG, granular layer þ/� þþ þþdorsal commissure, anterior þ � �dorsal fornix, anterior þþ þ/� �fimbra þ þ þ/�oriens layer þþþ þþþ þþþpyramidal cell layer þ þþþ þþ

Nucleus diagonal band

horizontal limb þþþ þþ þ/�vertical limb þþþ þþþ þ/�

Septum

lambdoid septal zone þþþþ þþ �lateral septal n., dorsal þþ þ �lateral septal n., intermediate þþþþ þþ þ/�lateral septal n., ventral þ þþþ þmedial septal n. þþþþ þþþ þ/�septofimbrial n. þþþ þþ þ/�septohippocampal n. þþþ þþ þ/�triangular septal n. þ þ/� þ

DiencephalonHypothalamus

anterior hypothalamic n. þ/� þ þ/�arcuate n. � þ �dorsomedial hypothalamic n. þ þ þlateral hypothalamic area þþþ þþ þlateroanterior hypothalamic n. þ/� þ �paraventricular hypothalamic n. þ þþþþ þþþperiventricular hypothalamic n. þ/� þþþ þþposterior hypothalamic area þþþþ þþ þpreoptic area, lateral þþ þþ þþpreoptic area, medial þ/� þ þ/�sub-paraventricular zone þ � �suprachiasmatic n. þ þ �supraoptic n. þþþ þþþ þþtuberomammillary n. þ þ þventromedial hypothalamic n. þ/þþ þ þ

Mammillary body

lateral mammillary n. þþ þ/� �medial mammillary þ � �premammillary n. þ þ þsupramammillary n. þþþ þþ þ/�

Thalamus

anterodorsal thalamic n. � þ þþþþcentromedial n. þ/� þ/� þ/�habenula þ þþþ þþþintermediodorsal thalamic n. þ � �Lateral posterior thalamic n. þ þ �n. access optic tract, dorsal terminal þþþþ þ �n. access optic tract, lateral terminal þþþþ þ �parafascicular thalamic n. � þ þþparaventricular thalamic n. þ þ/� þ/�peripeduncular n. þþþþ þþþþ þ/�periventricular fiber system � þþ �posterior intralaminar thalamic n. þþþ þþþ þ/�precommissural n. þ þþ �reuniens thalamic n. þ � þ/�rhomboid thalamic n. þ � þ/�subparafascicular thalamic n. � þþþ þþzona incerta þþ þ þ

Geniculate system

dorsolateral geniculate n. þ � �intergeniculate leaflet þþ þ/� �med geniculate n. þ � �subgeniculate n. þþ þ �suprageniculate n. þþ þ �ventrolateral geniculate n. þþþ þþ �



TABLE 1 (Continued)


Mesencephaloncaudal linear n. raphe þþþþ þþ þ/�cuneiform n. � þ �dorsal raphe n. þþþþ þþþ þinferior colliculus þ þþ �inferior colliculus, dorsal cortex � þþþ þþþintercollicular n. � þþ þinterfascicular n. þ þþþ �interpeduncular n. þþþ þþþ þþmed accessory oculomotor n. � þ �n. posterior commissure þ � �olivary pretectal n. þþ � �posterior commissure þþ � �posterior pretectal n. þþ þ �rostral linear n. raphe þ þ �substantia nigra þ � �substantia nigra, dorsal region þ (cb) � �supraoculomotor cap � þþ �ventral tegmental area þþ þþ þ/�Periaqueductal gray

anterior þþ þþ þanterior PAG, lateroventral region þ (cb) þþ þþdorsomedial þþþ þþ �dorsolateral þþþ þþþ þþlateral þþ þþ þ/�ventrolateral þþ þþ �

Superior colliculus

commissure þ þ �brachium þþ þ/� �deep gray layer þþ þþ þþdeep white layer þ þþ þþintermediate gray layer þþþ þþ þ/�intermediate white layer þþþ þ þ/�optic nerve layer þþ þþ þSuperficial gray þ þ þþþzona layer þ/� � þþþ

RhombencephalonBarrington’s n. þ þþ þcentral gray, pontine þþþ (cb) þþþ �inferior olive, medial þ þþþþ þþþþlateral lemniscus � þ þlateral parabrachial n. þ þþþ þlocus coeruleus þ/� � �medial vestibular n., parvicellular þþ (cb) þ þmedian raphe n. þþþþ þþ �n. incertus þþþþ (cb) þþ þþparamedian raphe n. þþþþ þþ �pontine raphe n. þþþ (cb) þ �prepositus hypoglossal n. þþ þ þraphe cap þþþ þ �raphe magnus þ/� þ þsolitary tract n. þ þþ þþspinal trigeminal n., dorsomedial � þþ þ/�spinal trigeminal n., interpolar � þ/� þTegmental system

anterior tegmental n. þþþ þþþ �dorsal tegmental n. þþ þþ þdorsomedial tegmental area þ þþþ �laterodorsal tegmental n., dorsal þ þþ þþlaterodorsal tegmental n., ventral � þ þþpedunculopontine tegmental n. þþ þþ �posterodorsal tegmental n. þ þ þventral tegmental n. þ þ �

Qualitative assessments of the distribution and relative densities of relaxin-3-LI, RXFP3 mRNA, and RXFP3 binding sites throughout the rostral-cau-

dal extent of the C57BL/6J mouse brain were made using the following scale: (�) no signal detected; (þ/�) scarce signal; (þ) low density of sig-

nal in the region; (þþ) moderate, (þþþ) high, and (þþþþ) very high density signal in the distinct nucleus/region. cb, cell bodies; n., nucleus.

Smith et al.


particularly enriched with high densities observed within

the dorsolateral region, while moderate densities were

detected in the anterior, dorsomedial, lateral, and ventro-

lateral regions, as well as the neighboring supraoculomo-

tor cap (Table 1). The superior colliculus was similarly

enriched, where two distinct bands of moderate density

RXFP3 mRNA expression were observed. The first was an

‘‘m-shaped’’ band that stretched the width of the superior

colliculus and included the optic nerve and intermediate

gray layers, while the second ‘‘straddled’’ the periaqueduc-

tal gray and included the deep gray and white layers (Fig.

5K,L). RXFP3 mRNA was also present in the inferior collicu-

lus, with high and moderate densities observed in the dor-

sal cortex (Fig. 11I) and adjoining posterior regions.

Other labeled regions include the interpeduncular nu-

cleus, with a high density of RXFP3 mRNA-positive cells

in the outer subnuclei (Fig. 11H), and the mesencephalic

raphe nuclei, where high and moderate densities were

detected in dorsal and caudal linear nuclei, respectively.

Finally, a high density was detected within the interfascic-

ular nucleus, and a moderate density was detected in the

intercollicular nucleus (Table 1).

RhombencephalonThe NI exhibited a moderate density of RXFP3 mRNA

(Fig. 5N), suggesting the existence of autoreceptors to

modulate the release of RLN3 from NI neurons. More lat-

erally, high and moderate densities were detected within

the central gray and lateral parabrachial nucleus, respec-

tively, while the locus coeruleus lacked RXFP3 mRNA

expression despite a moderate density present in the

neighboring area (possibly Barrington’s nucleus).

Various components of the tegmental system dis-

played RXFP3 expression, with high densities observed in

the anterior and dorsomedial regions, and moderate den-

sities detected in the pedunculopontine and dorsal

regions (Table 1). Interestingly, only low to moderate

RXFP3 mRNA expression was detected in the rhombence-

phalon raphe nuclei, including the raphe cap, median,

paramedian, and pontine raphe nuclei.

Caudal to the NI, low densities of RXFP3 mRNA-posi-

tive cells were present in the parvicellular medial vestibu-

lar and prepositus hypoglossal nuclei near the 4th ventri-

cle (Fig. 5O), and a moderate density was present in the

solitary tract and dorsomedial spinal trigeminal nucleus.

Finally, a very high density was present in the medial infe-

rior olive, the most caudal RXFP3-positive cells detected

in the mouse brain (Fig. 12N–P; Table 1).

Regional distribution of [125I]-R3/I5binding sites

In these experiments a low concentration (7–10 pM) of

[125I]-R3/I5 was used to determine the distribution of

RXFP3 binding sites in brain sections from WT and RLN3

KO/LacZ KI mice, and no discernable differences

between genotypes were detected. Relatively high levels

of nonspecific binding were observed in the presence of

100 nM unlabeled RLN3, but these were evenly distrib-

uted across brain sections (data not shown). Further-

more, due to the relatively low resolution inherent in x-ray

film, the regional sensitivity of this technique is below

that of those for the detection of RLN3 and RXFP3 mRNA.

Despite this, the present studies build upon earlier pre-

liminary experiments (Sutton et al., 2006); summarize the

distribution of [125I]-R3/I5 binding sites (Fig. 5), and pro-

vide semiquantitative scoring of the regional relative den-

sities (Table 1). Descriptions of this distribution through-

out the brain follow, with reference to relevant illustrative

autoradiograms.

[125I]-R3/I5 binding sites were most prominent within

the amygdala and hippocampus of the mouse brain (Fig.

5F–K). High to very high densities were observed in multi-

ple nuclei of the amygdala, including the lateral, medial,

basomedial, and basolateral subunits, in addition to the

amygdalohippocampal and amygdalopiriform transition

areas (Fig. 12C–E). In the hippocampus, binding sites were

enriched in the lateral and ventral regions, while more

medial regions displayed relatively sparse [125I]-R3/I5

binding sites (Fig. 12B–E). This resulted in an overall high

density of binding in the oriens layer and a moderate den-

sity of binding in the pyramidal cell layer and CA fields

(Table 1). In the dentate gyrus, high and moderate binding

was detected in the polymorph and granular layers,

respectively, while a low density was detected in the mo-

lecular layer (Table 1).

High to very high densities of [125I]-R3/I5 binding sites

were also observed in the bed nucleus of stria terminalis

(Fig. 12A), paraventricular hypothalamic nuclei (Fig.

12A,B), anterodorsal thalamic nucleus (Fig. 12B), habe-

nula (Fig. 12C), various subregions of the superior and in-

ferior colliculus (Fig. 12D; Table 1), and the inferior olive

(Fig. 5P).

Additionally, moderate densities of [125I]-R3/I5 binding

sites were detected in a range of other regions, including

the NI, supraoptic, lateral preoptic, and periventricular

hypothalamic nuclei (Fig. 12A), the interpeduncular nu-

cleus, subregions of the periaqueductal gray (Fig. 12E,F;

Table 1), the parafascicular and subparafascicular tha-

lamic nuclei (Fig. 12D), several tegmental subregions

(Table 1), and the nucleus of the solitary tract.

The distribution of [125I]-R3/I5 binding sites within the

cortex was of particular interest (Fig. 5B–K). Low, yet

readily detectable [125I]-R3/I5 binding was observed in a

mid-cortical ‘‘band’’ across the entire cortex at a level

equivalent to layer 4, which was absent from the more

medial regions of the cingulate and retrosplenial cortices



that lack this layer (Fig. 12A–E). Finally, a high density of

[125I]-R3/I5 binding sites was observed in the dorsal and

ventral endopiriform nuclei, which was only observable in

coronal sections collected close to bregma �0.94 mm

(Fig. 12B).

DISCUSSION

The present study employed a range of histological

techniques to produce the first comprehensive mapping

of the neuroanatomical distribution of RLN3 and its native

receptor, RXFP3, in the adult mouse brain. In situ hybrid-

ization of RLN3 mRNA, X-GAL histochemistry of a LacZ re-

porter gene in an RLN3 KO mouse and immunohisto-

chemical studies of RLN3-LI consistently revealed that

the major source of RLN3 expression in mouse brain is

large neurons that occupy the medial pontine NI, with

some scattered RLN3-positive neurons observed in the

adjacent pontine central gray and adjacent to the 4th

ventricle at more caudal levels; while separate small pop-

ulations of RLN3-positive neurons were detected in the

pontine raphe nucleus, anterior periaqueductal gray, and

a region dorsal to the substantia nigra and medial to the

peripeduncular nucleus. RLN3-LI was detected in nerve

axons, terminals, and boutons in a wide range of forebrain

areas, in a distribution characteristic of a transmitter/

neuromodulator. In general, a strong overlap was

observed between the distributions of the RLN3 nerve

fibers and RXFP3 mRNA and binding sites, in line with

RLN3 and RXFP3 representing a cognate ligand-receptor

pairing. Analysis of this distribution revealed several

RLN3/RXFP3-enriched brain regions and circuits associ-

ated with common or related functional modalities.

A majority of RLN3/RXFP3-rich brain regions can be

classified as functionally involved in ‘‘behavioral activa-

tion’’ (Goto et al., 2001; Swanson, 2003), and include the

septohippocampal, arousal, and sensory/visuospatial cir-

cuits. Notably, very high densities of RLN3/RXFP3 were

observed within the hippocampus and regions that regu-

late its function, including the medial septum/nucleus of

the diagonal band, posterior hypothalamus, and supra-

mammillary, interpeduncular, and median raphe nuclei,

which together constitute the septohippocampal pathway

(see e.g., Vertes, 1981; Vertes and Kocsis, 1997; Kirk,

1998; Pedemonte et al., 1998; Woodnorth et al., 2003;

Woodnorth and McNaughton, 2005; Jackson et al.,

2008). High levels of RLN3/RXFP3 were also observed in

arousal-related brain areas, such as the raphe nuclei, lat-

eral hypothalamus, and periaqueductal gray, while lower

levels were present in other arousal structures, such as

the ventral tegmental area and the pedunculopontine teg-

mental nucleus (see e.g., Mieda and Yanagisawa, 2002;

Saper et al., 2005; Lu et al., 2006; Monti and Jantos,

2008). Furthermore, RLN3 and/or RXFP3 were also

enriched in numerous structures involved in sensory/

visuospatial control, including the superior and inferior

colliculi, peripeduncular nucleus, lateral/dorsal terminals

of the nucleus of the optic tract, inferior olive, and cingu-

late and retrosplenial cortices (see e.g., Linke, 1999; Gib-

son et al., 2002; Vogt and Laureys, 2005; Dehmel et al.,

2008; Cappe et al., 2009; Halverson and Freeman, 2010).

Second, a number of RLN3/RXFP3-rich regions can be

classified as functionally involved in ‘‘stress responses,’’

and include hypothalamic and extrahypothalamic circuits

that play crucial roles in CRF mediated effects and regula-

tion of the hypothalamic–pituitary–adrenal (HPA) axis. A

large population of RXFP3-positive cells was observed

within the amygdala, while other positive stress- and anxi-

ety-related centers included the lateral septum, bed nu-

cleus of the stria terminalis, PVN, periaqueductal gray,

and dorsal raphe nucleus (see e.g., Potter et al., 1994;

Bittencourt and Sawchenko, 2000; Van Pett et al., 2000;

Justice et al., 2008; Lowry et al., 2008).

Comparative distribution of RLN3-LI, RXFP3mRNA, and [125I]-R3/I5 binding sites

In general, the distribution of RLN3-LI, RXFP3 mRNA,

and [125I]-R3/I5 binding sites appeared to overlap,

although some exceptions were noted. Such ‘‘mis-

matches,’’ which are commonly observed in ligand-recep-

tor systems (Herkenham, 1987; Justice et al., 2008), may

be due to several factors. For example, high densities of

RLN3-LI were observed in numerous raphe nuclei and the

cingulate and retrosplenial cortices, despite relatively low

densities of RXFP3 mRNA and binding sites, which may

indicate that RLN3-positive nerve fibers make few termi-

nal contacts in these regions, and instead project to more

distant structures. Conversely, regions including the

amygdala and paraventricular hypothalamic nucleus dis-

played relatively low densities of RLN3-LI and very high

densities of RXFP3 mRNA and binding sites, which apart

from technical difficulties of detection may indicate either

a high rate of local peptide release and relative depletion

of RLN3 from nerve terminals, and/or that RXFP3 present

in the amygdala and the paraventricular hypothalamic

nucleus are activated by RLN3 released from more dis-

tant sources, via dendritic (Justice et al., 2008) or volume

transmission (Zoli et al., 1999).

Furthermore, high levels of RXFP3 mRNA, but low lev-

els of [125I]-R3/I5 binding sites, were observed in regions

such as the septum and peripeduncular and posterior

intralaminar thalamic nuclei. This may reflect a lower sen-

sitivity and resolution of the binding assay or the reduced

[125I]-R3/I5 binding sites could result from high rates of

receptor internalization, turnover, and/or desensitization

Smith et al.


in ligand-rich regions, and high levels of endogenous

RLN3 binding to RXFP3 blocking [125I]-R3/I5 binding.

However, an explanation involving ligand regulation is not

supported by the observation that the distribution and rel-

ative densities of [125I]-R3/I5 binding were largely identi-

cal in RLN3 KO and WT mice (data not shown).

Interestingly, there were several instances where

bands of [125I]-R3/I5 binding sites were observed to be

differentially located to cells producing RXFP3 mRNA

within the same brain region, suggesting that within these

structures RXFP3 mRNA is produced in cell bodies and

the receptor protein is trafficked to the dendritic or nerve

terminal surface in a different anatomical location (layer/

region). For example, in the cerebral cortex RXFP3 mRNA

was expressed in neurons of the outer layers, whereas

low but robust [125I]-R3/I5 binding was restricted to a mid-

cortical ‘‘band’’ across the entire cortex at a level equiva-

lent to layer 4, which was absent from the more medial

regions of the cingulate and retrosplenial cortices. Also in

the superior colliculus, where the majority of RXFP3

mRNA-positive cells were within more internal layers,

[125I]-R3/I5 binding sites were more abundant peripherally

in the more dorsal zona and superficial gray layers.

Comparative analysis of RLN3/RXFP3distribution in different species

In addition to the highly conserved structure of RLN3

(Wilkinson et al., 2005), the enrichment of RLN3 in neu-

rons of the mouse NI further demonstrates strong homol-

ogy with the rat (Tanaka et al., 2005; Ma et al., 2007),

and with the nonhuman primate, as in a recent study high

levels of RLN3-LI were detected in a pontine region ho-

mologous to the rodent NI in the Macaca fascicularis (Ma

et al., 2009b). However, species differences will likely

exist in the distribution and neurochemistry of RLN3-posi-

tive and -negative neurons within the NI. For example,

based on its cytoarchitecture and efferent and afferent

connections, the rat NI has been defined as comprising a

densely packed medial pars compacta and a more diffuse

and lateral pars dissipata (Goto et al., 2001; Olucha-Bor-

donau et al., 2003). Due to the similar neuroanatomical

distribution of RLN3-positive neurons in the rat brain, it

has been postulated that RLN3 represents a useful

marker of the NI (Tanaka et al., 2005; Ma et al., 2007),

although there are clearly populations of RLN3-negative,

GABA neurons in the rat NI (Ma et al., 2007). An in-depth

anatomical and neurochemical analysis of the mouse NI

should reveal whether RLN3 is also a useful marker for

the NI in this species, or whether the mouse NI displays a

distinct morphology with a different proportion of RLN3-

positive and negative cells, particularly along the midline

(Franklin and Paxinos, 1997; Dong, 2008).

The presence of smaller numbers of RLN3 neurons in

the periaqueductal gray, pontine raphe, and in an area

dorsal to the substantia nigra and medial to the peripe-

duncular nucleus also demonstrates high homology with

the rat (Tanaka et al., 2005; Ma et al., 2007). Although

the contribution that each of these populations makes to

the forebrain pattern of RLN3 innervation is currently

unknown, it is likely that each population has differing

efferent and afferent connections, and may respond to

different local regulatory signals, as well as some stimula-

tory signals that activate the NI. Thus, further study of

these populations and their functional association with

each other and with neurons in the NI remains an impor-

tant step in elucidating the role of central RLN3/RXFP3

signaling.

In general, the distribution of RLN3 efferent projections

and RXFP3 expression/binding sites was very similar in

mice (current study; Sutton et al., 2006) and rats (Tanaka

et al., 2005; Ma et al., 2007), further supporting the exis-

tence of a conserved neuropeptide/receptor system.

However, some differences were apparent. For example,

the presence of RLN3-LI and RXFP3 in the substantia

innominata and the olivary and posterior pretectal nuclei

reported here in the mouse was not described in the rat,

whereas regions which may be more influenced by

RLN3/RXFP3 signaling in the rat than in the mouse

include the olfactory bulb, entorhinal cortex, arcuate

nucleus, and paraventricular thalamic nucleus. Analysis

of the distribution of RLN3 containing projections in the

M. fascicularis also revealed a potentially significant dif-

ference to rodents, as RLN3-LI was absent from midline

cortical regions of macaque brain (Ma et al., 2009b),

while high levels were observed in this region in mouse

and rat. The precise explanation for these differences is

currently unknown.

Putative functions of RLN3/RXFP3signaling in mouse brain

Although to date only a limited number of studies have

been conducted to investigate RLN3/RXFP3 function in

mice (Smith et al., 2009a; Sutton et al., 2009), based on

these and the neuroanatomical distribution of murine

RLN3/RXFP3, it is possible to speculate on the putative

functions of this system. As described above, high den-

sities of RLN3 and RXFP3 were observed in septohippo-

campal, arousal, and sensory/visuospatial control cir-

cuits, indicating that RLN3/RXFP3 signaling likely

modulates behavioral activation/control (Goto et al.,

2001; Swanson, 2003). Specifically, one of the major

functions of the septohippocampal pathway is the gener-

ation/modulation of hippocampal theta rhythm, which is

considered the ‘‘on’’ state of this important structure



involved in vigilance, exploratory behavior, and related

tasks such as navigation and working memory (Vertes

and Kocsis, 1997; Vertes, 2005; Villarreal et al., 2007).

Further to the demonstration that NI stimulation

increased hippocampal theta rhythm in anesthetized rats

(see introduction), we have recently shown that injection

of the RXFP3 agonist, R3/I5, into the medial septum

increased hippocampal theta rhythm, and that this effect

was blocked by pretreatment with an RXFP3 antagonist

(Ma et al., 2009a). These studies also demonstrated

effects of endogenous RXFP3 signaling in the medial sep-

tum on theta activity in conscious rats and on spatial

memory (Ma et al., 2009a).

Furthermore, central administration (i.c.v.) of R3/I5 in

rats produced a dose-related increase in locomotor activ-

ity in automated locomotor cells (Sutton et al., 2009),

while in a study of mixed-background RLN3 KO mice,

they displayed hypoactivity in multiple behavioral para-

digms compared to their WT littermates (Smith et al.,

2009a). These effects on locomotor behavior are in line

with the presence of RLN3/RXFP3 in brain regions associ-

ated with arousal such as the median raphe, lateral hypo-

thalamus, periaqueductal gray, ventral tegmental area,

tuberomammillary, and pedunculopontine tegmental nuclei;

however, interactions with arousal-related transmitters

such as orexin, which are expressed by subpopulations of

neurons within these regions are yet to be investigated.

In addition to arousal, the distribution of RLN3/RXFP3

in the mouse brain supports the idea that RLN3/RXFP3

signaling modulates stress responses through interac-

tions with CRF. Two of the most prominent regions that

displayed RXFP3 expression/binding sites were the

amygdala (Gray and Bingaman, 1996) and PVN (Herman

et al., 2002), while many other structures commonly

linked with stress responses also displayed robust

RLN3/RXFP3, such as the bed nucleus of the stria termi-

nalis (Lee et al., 2008), lateral septum (Henry et al.,

2006), periaqueductal gray (Bowers et al., 2003), and the

dorsal/median raphe nuclei (Graeff et al., 1996; Lowry

et al., 2008; Ohmura et al., 2008). A role of RLN3/RXFP3

in stress responses is further supported by the observed

activation of RLN3 neurons within the NI following neuro-

genic stressors and i.c.v. administration of CRF in the rat

(Tanaka et al., 2005; Banerjee et al., 2010), and the pres-

ence of a moderate density of CRF1 in the mouse NI (Van

Pett et al., 2000; Justice et al., 2008). However, as stress-

related regions such as the parvocellular PVN contain

populations of CRF-positive and -negative neurons (Her-

man et al., 2002), identifying the neurochemical pheno-

type of the cells that express RXFP3 within these regions

will be an important focus of future research.

Finally, the presence of high densities of RXFP3 in the

periventricular hypothalamic nucleus and magnocellular

PVN and supraoptic nucleus suggests a role for RLN3/

RXFP3 signaling in aspects of neuroendocrine and meta-

bolic control in mice. In particular, a possible modulation

of feeding behavior is predicted in light of the robust

increases in food intake and body weight elicited by cen-

tral infusion of relaxin-3 in rats (McGowan et al., 2005,

2006, 2007; Hida et al., 2006). In line with this, a cohort

of mixed background RLN3 KO mice displayed reduced

body weight, body fat, and plasma leptin and insulin lev-

els relative to WT controls when fed on a high fat diet

(Sutton et al., 2009). These findings have not been con-

firmed in backcrossed cohorts, however (Sutton S.W.,

unpubl. data), suggesting that further research is required

in this area. It is also unclear what proportion of the

increased feeding behavior observed in rats was driven

by actions of RLN3/RXFP3 signaling on arousal, as

reported for the peptide transmitters orexin (Willie et al.,

2001; Mieda and Yanagisawa, 2002) and melanin-con-

centrating hormone (Huang et al., 2007), and the mono-

amine histamine (Valdes et al., 2005).

CONCLUSIONS

These anatomical findings further support the con-

served native pairing of RLN3 and RXFP3 in mammals

and provide evidence for broad modulatory actions of

RLN3/RXFP3 networks. The main source of RLN3 in the

mouse is the pontine NI, and RLN3-positive projections

and RXFP3-positive cells are enriched in septohippocam-

pal, arousal, and sensory/visuospatial control circuits

associated with behavioral activation, and in hypothala-

mic and limbic circuits linked to stress and affective

responses. Our findings should assist the design and

interpretation of future anatomical studies to identify

RLN3 target neurons, pharmacological studies of the

effects of RXFP3 agonist and antagonist peptides on

mouse physiology and behavior, and studies to determine

the biological consequences of RLN3 deficiency in KO

mice (Smith et al., 2006a, 2009a,b). It is of importance to

determine the neurochemical identity of RXFP3-positive

cells in different brain regions, as this will provide further

insights into the function of endogenous RLN3/RXFP3

signaling. In this respect, we are currently attempting to

develop robust methods to histologically label RXFP3

using selective RXFP3 antibodies and/or reporter gene

mouse strains (e.g., Gould and Zingg, 2003; Liu et al.,

2003c), including a bacterial artificial chromosome trans-

genic mouse line in which the distribution of RXFP3 can

be conveniently tracked by assessing the distribution of a

fluorescent marker protein (Gong et al., 2003; Justice

et al., 2008). While the putative functional interactions

predicted by the observed neuroanatomical framework

are speculative, several are supported by existing findings

Smith et al.


in rats and represent proposed actions that are testable

experimentally in mice. Such studies should better estab-

lish the role of RLN3/RXFP3 networks in the mammalian

brain and identify their therapeutic potential in the treat-

ment of neurological and psychiatric diseases.

ACKNOWLEDGMENTS

This article is dedicated to Prof. Geoffrey Tregear on

the occasion of his retirement as Deputy Director of the

Howard Florey Institute. The authors thank Tania Ferraro

for assistance with the preparation of antisera used in

these studies. During these studies C.M.S. was the recipi-

ent of an NHMRC (Australia) Dora Lush Biomedical Post-

graduate Scholarship.

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Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain