Neuroscience 144 (2007) 165190
RELAXIN-3 IN GABA PROJECTION NEURONS OF NUCLEUS
INCERTUS SUGGESTS WIDESPREAD INFLUENCE ON FOREBRAIN
CIRCUITS VIA G-PROTEIN-COUPLED RECEPTOR-135 IN THE RAT
S. MA,a* P. BONAVENTURE,d T. FERRARO,a
P.-J. SHEN,a T. C. D. BURAZIN,a R. A. D. BATHGATE,a,b
C. LIU,d G. W. TREGEAR,a,b S. W. SUTTONd
AND A. L. GUNDLACHa,c
a
Howard Florey Institute, The University of Melbourne, Melbourne,
Victoria 3010, Australia
b
Department of Biochemistry and Molecular Biology, The University of
Melbourne, Melbourne, Victoria, Australia
c
Department of Anatomy and Cell Biology, The University of Mel-
bourne, Melbourne, Victoria, Australia
d
Neuroscience Group, Johnson & Johnson Pharmaceutical Research
and Development, LLC, San Diego, CA, USA
AbstractRelaxin-3 (RLX3) is a newly identified member of
the relaxin/insulin peptide family that is highly conserved
across a range of species from fish to mammals and is highly
expressed in rat, mouse and human brain. Extensive phar-
macological studies have demonstrated that RLX3 is a high
affinity, selective ligand for G-protein-coupled receptor-135
(GPCR135, now classified as relaxin family peptide-3 recep-
tor; RXFP3). In ongoing studies to understand the physiolog-
ical functions of RLX3, the distribution of RLX3-containing
neuronal elements in rat brain was determined by immuno-
histochemistry, using an affinity-purified polyclonal anti-
serum raised against a conserved segment of the RLX3 C-
peptide (AS-R385-101). Consistent with the distribution of
RLX3 mRNA, neurons containing RLX3-like immunoreactivity
(LI) were observed in the pontine nucleus incertus and the
majority of these cells, which are known to express cortico-
tropin-releasing factor receptor-1, were shown to express
glutamic acid decarboxylase-65-immunoreactivity, suggest-
ing a GABA phenotype. Nerve fibers and terminals containing
RLX3-LI were observed adjacent to cells in the nucleus in-
certus and in various forebrain regions known to receive
afferents from the nucleus incertus, including cortex, sep-
tum, hippocampus, thalamus, hypothalamus and midbrain.
Regions that contained highest densities of RLX3-positive
fibers included the medial septum, lateral preoptic area, lat-
eral hypothalamus/medial forebrain bundle and ventral hip-
pocampus; and additional fibers were observed in olfactory
bulb and olfactory and frontal/cingulate cortices, bed nucleus
of the stria terminalis, dorsal endopiriform, intergeniculate,
*Corresponding author. Tel: 61-3-8344-6759; fax: 61-3-9347-0446.
E-mail address: s.ma@hfi.unimelb.edu.au (S. Ma).
Abbreviations: CRF, corticotropin-releasing factor; CRF-R1, cortico-
tropin-releasing factor receptor-1; DAB, 3,3=-diaminobenzidine; DR,
dorsal raphe nucleus; GAD65, glutamic acid decarboxylase-65;
GPCR135, G-protein-coupled receptor-135; GPCR142, G-protein-
coupled receptor-142; IGL, intergeniculate leaflet; LH, lateral hypothal-
amus; -LI, -like immunoreactivity; LPO, lateral preoptic area; LS, lat-
eral septum; MPO, medial preoptic area; MS, medial septal nucleus;
NDB, nucleus of the diagonal band; NHS, normal horse serum; NI,
nucleus incertus; NIc, nucleus incertus compacta; NId, nucleus incer-
tus dissipata; PP, peripeduncular nucleus; PVN, paraventricular nu-
cleus; RLX3, relaxin-3; VGlut2, vesicular glutamate transporter-2.
0306-4522/07$30.000.00 2006 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2006.08.072
165
and supramammillary nuclei, and the periaqueductal gray
and dorsal raphe. The RLX3-positive network overlapped the
regional distribution of GPCR135 mRNA and specific binding
sites for an [125I]-GPCR135-selective, chimeric peptide. These
anatomical findings further support the proposition that
RLX3 is the endogenous ligand for GPCR135 in rat brain and
provide evidence for broad modulatory activity of RLX3 in
behavioral activation relating to autonomic and neuroendo-
crine control of metabolism and reproduction and higher-
order processes such as stress and cognition. 2006 IBRO.
Published by Elsevier Ltd. All rights reserved.
Key words: relaxin-3 like-immunohistochemistry, GPCR135
mRNA, nucleus incertus efferents, stress, septo-hippocam-
pal theta rhythm.
The insulin/relaxin peptide superfamily consists of a large
number of peptides expressed in a wide range of species
(Wilkinson et al., 2005; Bathgate et al., 2006a). Relaxin-3
(RLX3), first identified in 2002 by our laboratory (Bathgate
et al., 2002; Burazin et al., 2002), is the latest addition to
this family; and in contrast to relaxin that is expressed in
several tissues, especially the female reproductive tract,
RLX3 is primarily expressed in the brain of human, mouse
and rat (Bathgate et al., 2002, 2003; Burazin et al., 2002).
In the rodent brain, RLX3 mRNA is enriched in a midline
pontine region embedded in the caudal ventromedial cen-
tral gray known as the nucleus incertus (NI) (Bathgate et
al., 2002; Burazin et al., 2002), which is a distinct cell group
that consists of two parts, the midline compacta (NIc) and
the dissipata (NId) that extends laterally from the NIc (Goto
et al., 2001; Olucha-Bordonau et al., 2003). Renewed
interest in the anatomy and physiology of the NI was
initially aroused by its high level of expression of cortico-
tropin-releasing factor receptor-1 (CRF-R1) mRNA, and a
belief that it might mediate some of the extra-pituitary
actions of CRF in response to stress (Potter et al., 1994;
Bittencourt and Sawchenko, 2000). Resultant anterograde
and retrograde tract-tracing studies of the NI suggested
that this nucleus is well positioned to integrate information
relating to memory and attentional state (Goto et al., 2001;
Olucha-Bordonau et al., 2003) and concurrent in vivo stud-
ies in rats reported that stressors such as forced swimming
or restricted food access increased Fos expression in NI
neurons (Goto et al., 2001, 2005).
Extensive genetic, molecular and pharmacological
studies have now shown that the preferred native receptor
for RLX3 is G-protein-coupled receptor-135 (GPCR135 or
somatostatin- and angiotensin-like peptide receptor
(SALPR); now classified as relaxin family peptide-3 recep-
166 S. Ma et al. / Neuroscience 144 (2007) 165190
tor; RXFP3 (Matsumoto et al., 2000; Liu et al., 2003a,b;
see Bathgate et al., 2006b). In separate studies of orphan
GPCRs, Liu et al. (2003b) isolated RLX3 from brain tissue
extracts and demonstrated that it potently bound and ac-
tivated the cloned GPCR135 in vitro; and to a lesser extent
the related receptor, G-protein-coupled receptor-142
(GPCR142) (or GPR100) (Liu et al., 2003a). In contrast,
relaxin, did not bind or activate either GPCR135 or
GPCR142 (Liu et al., 2003a,b); instead preferring leucine-
rich repeat-containing G-protein-coupled receptor-7 (LGR7)
(Hsu et al., 2002; Sudo et al., 2003). The GPCR142 and
INSL5 genes were subsequently found to be absent from
the rat genome and a pseudogene, respectively (Liu et
al., 2003a; Wilkinson et al., 2005), suggesting that INSL5 is
the cognate ligand for GPCR142 in mouse and human.
This possibility was strongly supported by pharmacological
studies that clearly demonstrated synthetic human INSL5
to be a specific and potent ligand at human GPCR142 and
expression data revealing high levels of both GPCR142
and INSL5 mRNA in tissues such as the colon (Liu et al.,
2005b). In contrast, GPCR142 and INSL5 mRNA are ef-
fectively undetectable in mouse brain and INSL5 does not
interact with specific RLX3 binding sites in rat brain sec-
tions, further supporting the RLX3GPCR135 pairing in
mammalian brain (Sutton et al., 2006). Functional activa-
tion of GPCR135 in vitro results in inhibition of cAMP via
Gi/o-protein coupling (Liu et al., 2003b).
Preliminary in situ hybridization histochemistry studies
have identified the regional distribution of GPCR135
mRNA in rat brain (Liu et al., 2003b; Sutton et al., 2004),
which is reflected by the distribution of specific binding
sites labeled by a chimeric peptide (RLX3 B-chain/INSL5
A-chain; [125I]-R3/I5) that is selective for GPCR135 (Sutton
et al., 2004; Liu et al., 2005a). In turn, this pattern of
GPCR135 expression is consistent with the widespread
projections of the NI to numerous forebrain regions re-
vealed in anterograde tract-tracing studies (Goto et al.,
2001; Olucha-Bordonau et al., 2003). During the course of
the current studies, Tanaka and colleagues (2005) re-
ported the existence of a network of RLX3-immunoreactive
fibers in rat brain, using a monoclonal antibody against the
N-terminus of RLX3 and electron microscopic analysis
revealed RLX3-immunoreactivity present in dense-core
vesicles in the perikarya and synaptic terminals of axons in
the hypothalamus (Tanaka et al., 2005), consistent with
release of RLX3 as a transmitter/modulator.
In this study, using an affinity-purified polyclonal anti-
serum raised against a region of the RLX3 C-peptide that
is homologous in human, rat and mouse, we examined the
distribution of RLX3-LI throughout the rat brain, and inves-
tigated the nature of the primary transmitter used by RLX3-
containing neurons in the NI in double-label immunofluo-
rescence experiments. We also further documented the
relative correlation between the distribution of RLX3-posi-
tive neural elements and GPCR135 mRNA in various cor-
tical and sub-cortical circuits (Liu et al., 2003b; Sutton et
al., 2004), in relation to potential functional associations.
Preliminary reports of some of these findings have been
published in abstract form (Banerjee et al., 2005; Ma et al.,
2006).
EXPERIMENTAL PROCEDURES
Animals
Experiments described were carried out with the approval of the
Howard Florey Institute Animal Welfare Committee and according
to ethical guidelines issued by the National Health and Medical
Research Council of Australia. All efforts were made to mini-
mize the number of animals used and their suffering. Male
SpragueDawley rats (n12; 250 300 g) were obtained from
the Australian Research Centre (Canning Vale, WA, Australia)
and maintained on a 12-h light/dark cycle. Rats were killed by
halothane inhalation and perfused transcardially with 100 ml
ice-cold PBS solution (PBS: 137 mM NaCl, 2.7 mM KCl,
11.2 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) followed by
300 ml ice-cold 4% paraformaldehyde in PBS (pH 7.4) and
then decapitated. Brains were dissected and immersed in fix-
ative for 1 h at 4 C before cryoprotection in 20% sucrose in
PBS overnight at 4 C. Coronal or parasagittal sections (40 m)
were cut on a cryostat at 16 C (Cryocut 1800, Leica Micro-
systems, Heerbrugg, Switzerland) and collected into PBS im-
mediately before immunohistochemical processing.
For some studies (incl. glutamic acid decarboxylase-65
(GAD65) and vesicular glutamate transporter-2 (VGlut2) immuno-
histochemistry), rats were given a bilateral injection of colchicine
into the lateral ventricles 24 h prior to perfusion, to promote the
accumulation of transmitters and/or enzymes in neuronal somata.
Briefly, rats were anesthetized using an isoflurane inhalation mask
(2% at 0.2 l/min), the skull was positioned in a stereotaxic frame
(Kopf Instruments, Tujunga, CA, USA) and holes were drilled
through the skull [coordinates: anteroposterior, 0.2 mm from
bregma; mediolateral, 1.5 mm from midline; dorsoventral,
4.0 mm from skull surface]. Bilateral injections of colchicine
(3.5 l of a 10 g/ml solution) were made with a 10 l Hamilton
syringe (Harvard Apparatus, Holliston, MA, USA) via polyethylene
tubing connected to a 30-gauge needle. The injection needle was
left in place for a further 60 s to maximize colchicine diffusion
before it was withdrawn.
Antibody production
Polyclonal antisera were raised in rabbits against a synthetic
peptide equivalent to amino acid residues 85-101 of the RLX3
pro-peptide. These residues are contained in the C-peptide region
and are identical in mouse and human with one conservative
amino acid change from the rat sequence (Fig. 1) (Bathgate et al.,
2002; Burazin et al., 2002). The peptide was conjugated through
a C-terminal cysteine residue to keyhole limpet hemocyanin and
injected s.c. in rabbits with Freunds complete adjuvant. Bleeds
were tested using dot blotting for immunoreactivity with the pep-
tide epitope as well as mouse pro-RLX3 (Bathgate et al., 2006b).
One of three rabbits that displayed significant antibody production
was exsanguinated and collected plasma containing antisera was
purified using an affinity-column. Affinity purified AS-R385-101 was
utilized at a final dilution of 1:2500 (see below). The specificity of
this antiserum was tested by incubating a 1:2500 dilution for 24 h
at 4 C with 1 g/ml of mouse pro-RLX3 before incubation with
brain sections (see below).
Immunohistochemistry
Free-floating sections were incubated in 1% H2O2 in 70% meth-
anol for 30 min with gentle shaking, rinsed 35 min in PBS, and
then incubated in blocking buffer (10% (v/v) normal horse serum
(NHS) and 0.3% (v/v) Triton X-100 in PBS) for 1 h with shaking at
room temperature. Sections were then incubated in PBS contain-
 S. Ma et al. / Neuroscience 144 (2007) 165190 167

Mouse 1 MAMLGL-LLLASWALLGALGLQAEARPAPYGVKLCGREFIRAVIFTCGGSRWRRADILAH
Rat 1 MATRG--LLLASWALLGALVLQAEARPAPYGVKLCGREFIRAVIFTCGGSRWRRADILAH
Human 1 MARYMLLLLLAVWVLTGELWPGAEARAAPYGVRLCGREFIRAVIFTCGGSRWRRSDILAH

Mouse 60 ESLGDFFADGEANTDHLASELDEAVGSSEWLALTKSPQAFYGGRASWQGSPGVVRGSRDV
Rat 59 DPLGEFFADGEANTDHLASELDEAVGSSEWLALTKSPQVFYGGRSSWQGSPGVVRGSRDV
Human 61 EAMGDTFPDADADEDSLAGELDEAMGSSEWLALTKSPQAFYRGRPSWQGTPGVLRGSRDV

Mouse 120 LAGLSSSCCEWGCSKSQISSLC

Rat 119 LAGLSSSCCEWGCSKSQISSLC
Human 121 LAGLSSSCCKWGCSKSEISSLC

Fig. 1. Amino acid sequence of pro-RLX3 of mouse, rat and human. Conserved amino acids are shaded. AS-R385101 was generated against a
conserved region of the C-peptide, with one amino acid change from the rat sequence (box).

ing polyclonal RLX3 antiserum (AS-R385-101 dilution 1:2500), 2%
NHS, 0.3% Triton X-100, and 0.1% NaN3 (pH 7.4) for 72 h at
4 C. Sections were washed 35 min in PBS and then incubated
in a 1:500 dilution of secondary biotinylated anti-rabbit IgG (Vector
Laboratories, Burlingame, CA, USA) with constant stirring for 1 h
at room temperature, followed by 35 min washes. Sections were
incubated in a 1:100 avidin biotin complex solution (Vectastain
Elite, Vector Laboratories) in PBS for a further 45 min at room
temperature, followed by 35 min washes. Immunostaining was
visualized using a standard peroxidase chromogen reaction by
placing the sections in 0.5 mg/ml 3,3=-diaminobenzidine (DAB) in
PBS (Sigma-Aldrich, Castle Hill, NSW, Australia) followed by ad-
dition of 0.06% H2O2 for a further 2 6 min. Sections were washed
for 35 min and mounted onto gelatin-chrom alum-coated glass
microscope slides and left to dry overnight. Sections were then
dehydrated and cleared through a series of ethanol and xylene
and coverslipped using DPX mounting media (Sigma-Aldrich).
Pre-absorption with 1 g/ml pro-RLX3 C-peptide at 4 C for 24 h,
completely abolished specific immunostaining of fibers observed
within the medial septum and no staining was observed in sec-
tions incubated with pre-immune serum or when no primary anti-
serum was present in the incubation (Fig. 2).
For fluorescence microscopy, sections were incubated with
anti-RLX3 (dilution 1:1000) and either monoclonal anti-GAD65
(1:500; NICHD Developmental Studies Hybridoma Bank, The Uni-
versity of Iowa, Department of Biological Sciences, Iowa City, IA,
USA), guinea-pig anti-VGlut2 (1:1000; Chemicon International
Inc., Temecula, CA, USA), or monoclonal anti-calbindin D-28K
(1:4000; Sigma-Aldrich), overnight at 4 C, followed by 35 min
washes. Sections were then incubated for at least 2 h with Al-
exa549-conjugated goat anti-rabbit IgG (1:250; Invitrogen Corpo-
ration, Sydney, NSW, Australia) for detection of anti-RLX3; with
biotinylated anti-guinea-pig IgG followed by FITC-conjugated avi-
din (Jackson ImmunoResearch Laboratories Inc., West Grove,
PA, USA) for detection of anti-VGlut2; and with biotinylated anti-
mouse IgG (Vector Laboratories) followed by FITC-conjugated
avidin for detection of anti-GAD65 or anti-calbindin.
In situ hybridization autoradiography
As previously described (Sutton et al., 2004), coronal sections
(20 m) of rat brain were cut using a Cryostat microtome (Microm
HM505E, Mikron, San Diego, CA, USA), thaw-mounted on adhe-
sive microscope slides (Superfrost Plus; VWR, San Diego, CA,
USA) and stored at 70 C until use. Adjacent sections were used
for in situ hybridization and radioligand binding autoradiography
(see below). Transcription of a full length rat GPCR135 cDNA
probe (GenBank accession No. AY633763) was accomplished
using the Promega Riboprobe Combination T3/T7 kit and [35S]-
UTP (Perkin Elmer, Boston, MA, USA). Sections were fixed for 30
min at 4 C using 4% paraformaldehyde, pH 9.5; washed in
Dulbeccos phosphate-buffered saline; treated with 3 g/ml pro-
teinase K (Sigma-Aldrich, St. Louis, MO, USA) and acetylated with
1:400 acetic anhydride in 0.1 M triethanolamine pH 8.0; rinsed in
2SSC; dehydrated through an alcohol series and dried for 2 h in
a vacuum desiccator at room temperature. Slides were covered
with hybridization solution and glass coverslips were applied and
sealed with DPX mountant (Fluka, Milwaukee, WI, USA) prior to
incubation at 60 C. Following the stringency wash, slides were
washed in 0.1SSC; dehydrated through an ethanol series and
then dried in a vacuum desiccator. Slides were exposed to X-ray
film (Kodak Biomax MR, Rochester, NY, USA) for 4 weeks; then
processed using a Konica SRY-101A film processor (Konica Med-
ical Corporation, Wayne, NJ, USA).
Radioligand-binding autoradiography
As previously described, brain sections were thawed and dried
under a cold air stream and then pre-incubated for 15 min 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) (Sutton
et al., 2004). Sections were dried again under a cold air stream
and incubated for 60 min with 7 pM [125I]-R3/I5 (specific activity,
2200 Ci/mmol) in incubation buffer containing 0.5% bovine serum
albumin and protease inhibitor cocktail. Non-specific binding was
determined in the presence of 100 nM human RLX3. After incu-
bation, excess radioligand was removed by immersing slides in
incubation buffer at 4 C (310 min) followed by a quick immer-
sion in water. Sections were dried and exposed to Kodak Omat
film for 8 weeks; then developed and fixed in a Konica SRY-101A
film processor.
Photography and image production
Immunoperoxidase-stained brain sections were examined under
bright- and dark-field illumination on a Nikon Microphot SA micro-
scope (FSE, Melbourne, VIC, Australia) and digital images were
acquired using a Sony CCD camera and MCID-M2 software (Im-
aging Research Inc., St Catharines, ON, Canada). Immunofluo-
rescence was analyzed using an Olympus IX70 microscope
(Olympus Imaging, Melbourne, VIC, Australia) equipped with a
SPOT real-time digital camera with Image-pro 4.5 software (Di-
agnostics Instruments, Sterling Heights, MI, USA) and a BioRad
MRC1024 confocal scanning laser system on a Zeiss Axioplan 2
microscope equipped with an argon krypton laser (BioRad, Rich-
mond, CA, USA). Samples were scanned sequentially to collect
light emitted as red or green fluorescence using 63 and 100
objectives and 1.4 zoom. Fluorescence was quantified using the
168 S. Ma et al. / Neuroscience 144 (2007) 165190

Abbreviations used in the figures

ac anterior commissure LRt lateral reticular n

aca anterior commissure, anterior LS lateral septum
Acb accumbens n LSD lateral septal nucleus, dorsal
aci anterior commissure, intrabulbar LSI lateral septal nucleus, intermediate
AHi amygdalohippocampal area LSV lateral septal nucleus, ventral
alv alveus of the hippocampus LV lateral ventricle
AOB accessory olfactory bulb mfb medial forebrain bundle
AOD anterior olfactory n, dorsal MGV medial geniculate n, ventral
AOL anterior olfactory n, lateral mlf medial longitudinal fasciculus
AOM anterior olfactory n, medial MM medial mammillary n, medial
AOV anterior olfactory n, ventral MnPO median preoptic n
Aq aqueduct MPA medial preoptic area
asc7 ascending fibers of facial nerve MS medial septum
BLA basolateral amygdala MTU medial tuberal n
BMA basomedial amygdala NDB n diagonal band
BNST bed n of stria terminalis NI nucleus incertus
BSTLD bed n of stria terminalis, lateral division, dorsal NIc nucleus incertus compacta
BSTLV bed n of stria terminalis, lateral division, ventral NId nucleus incertus dissipata
BSTM bed n of stria terminalis, medial division Op optic nerve layer, superior colliculus
BSTMA bed n of stria terminalis, medial division, anterior opt optic tract
CA1-3 CA13 fields of hippocampus ox optic chiasm
Cb cerebellum PAG periaqueductal gray
cc corpus callosum PDTg posterodorsal tegmental n
CC central canal Pf parafascicular thalamic n
CEA central amygdala PH posterior hypothalamic area
cg cingulum Pir piriform cortex
Cg cingulate cortex Pn pontine reticular n
CGPn central gray of pons PnC pontine reticular n, caudal
Cl claustrum
PoDG polymorph layer of the dentate gyrus
CL centrolateral thalamic n
PP peripeduncular n
CM centromedial thalamic n
PrH prepositus hypoglossal n
CPu caudate putamen
PV paraventricular thalamic n
DCIC dorsal cortex, inferior colliculus
PVA paraventricular thalamic n, anterior
DG dentate gyrus of hippocampus
PVN paraventricular hypothalamic n
DLG dorsal lateral geniculate n
Re reuniens thalamic n
DMH dorsomedial hypothalamic n
Rh rhomboid thalamic n
DR dorsal raphe n
RPn raphe pontis n
DTg dorsal tegmental n
RRF retrorubral field
ec external capsule
Ent entorhinal cortex RSA retrosplenial agranular cortex
E/OV ependymal and sub-ependymal layer/olfactory RSG retrosplenial granular cortex
ventricle SC superior colliculus
EPd dorsal endopiriform n scc splenium of corpus callosum
EPl external plexiform layer of olfactory bulb SFi septofimbrial n
f fornix SHy septohypothalamic n
fi fimbria of hippocampus sm stria medullaris of thalamus
fmi forceps minor of corpus callosum SN substantia nigra
g7 genu of facial nerve sol solitary tract
Gr granule cell layer of olfactory bulb SPTg subpeduncular tegmental n
Gl glomerular layer of olfactory bulb st stria terminalis
Hb habenula n Sp5 spinal trigeminal n
HDB n of horizontal limb of diagonal band Sub subiculum
ic internal capsule SuG superficial gray layer, superior colliculus
ICjm islands of Calleja, major SuM supramammillary n
IF interfascicular n tfp transverse fibers of the pons
IGL intergeniculate leaflet TS triangular septal n
IGr internal granule layer of olfactory bulb TT tenia tecta
IP interpeduncular n VDB n of vertical limb of diagonal band
IPC interpeduncular n, caudal vhc ventral hippocampal commissure
IPDL interpeduncular n, dorsolateral VLG ventral lateral geniculate n
IPI interpeduncular n, intermediate VMH ventromedial hypothalamic n
IPl internal plexiform layer of olfactory bulb VP ventral pallidum
IPL interpeduncular n, lateral VTA ventral tegmental area
IPR interpeduncular n, rostral xscp decussation of the superior cerebellar peduncle
LDTg laterodorsal tegmental n 3V third ventricle
LH lateral hypothalamic area 4V fourth ventricle
LPG lateral paragigantocellular n 12 hypoglossal n
LPO lateral preoptic area hypothalamus
 S. Ma et al. / Neuroscience 144 (2007) 165190
Fig. 2. Coronal sections through the septum incubated in (A) polyclonal anti-RLX3 antiserum (AS-R385101) only, (B) overnight preabsorption of
antiserum with 1 g/ml of rat RLX3 C-peptide, (C) pre-immune serum, and (D) antibody diluent only. Inset: high resolution images of the medial
septum. Immunoreactive fibers were detected with the antiserum, however no immunoreactive elements were observed following preabsorption,
pre-immune serum, or absence of primary antibody. Scale bars600 m (AD) and 300 m (inset).
NIH-Image software program (National Institutes of Health, Be-
thesda, MD, USA).
Digital images from X-ray film autoradiograms were captured
using MCID-M2 software (Imaging Research Inc.) via a Sony
XC-77 camera (Berthold Australia, Bundoora, VIC, Australia).
Brightness and contrast levels of these images were adjusted and
composite plates were compiled and labeled using Adobe Photo-
shop 7.0 for Macintosh OSX (Adobe Systems Inc., San Jose,
CA, USA). All micrographs were archived as high-resolution im-
ages (500 dpi) in Photoshop CS (Adobe) and after any required
cropping, adjustment for contrast or removal of obvious artifacts;
plates were compiled and labeled using Photoshop CS for
Macintosh OSX (Adobe). Schematics illustrating the distribution of
RLX3-LI were created using Brain Maps: Structure of the Rat
Brain software (Swanson, 1998, 1999) and Photoshop CS for
Macintosh OSX (Adobe).
RESULTS
Specificity of RLX3 antiserum
In the current study, the distribution of RLX3-like immuno-
reactivity (-LI) was investigated using an affinity-purified
polyclonal antiserum raised against a short 16 amino acid
sequence of the C-peptide that is identical in the RLX3
sequences of mouse, rat, and human (Bathgate et al.,
2002; Burazin et al., 2002) and the specificity of the immu-
nostaining with AS-R385-101 was assessed in various
ways. In nave and colchicine-treated rats, the cellular
distribution of RLX3-LI was compatible with that of RLX3
mRNA (Burazin et al., 2002; Liu et al., 2003b; Tanaka et
al., 2005) and the immunoreaction in the NI and septum
was completely blocked by pre-incubation of the antiserum
with low micromolar concentrations of the peptide antigen
(AS-R385-101; Figs. 1 and 2; data not shown). Only broad,
non-specific cellular staining was observed in sections
incubated with pre-immune sera. Furthermore, no specific
staining was observed in sections processed with primary
antiserum excluded from the incubation. In a further im-
portant test, specific immunostaining was absent in brain
sections from RLX3 knock-out mice brains (Smith CM, Ma
S, Sutton SW, Gundlach AL, unpublished observations).
Previous studies with an antiserum raised against full-
length relaxin (R6), showed positive staining within the
NI, suggesting overlap with antigenic sequences com-
mon to both peptides (Burazin et al., 2002), while in
contrast, cells previously shown to contain relaxin-LI
and relaxin mRNA (Ma et al., 2005) were not labeled
169
with the RLX3 antiserum used in the current study.
Lastly, although the C-peptide directed antiserum does
not directly recognize the mature A/B chain form of
RLX3, the results observed correlate well with those
observed in a recent study using a monoclonal antibody
raised against the N-terminal of the A-chain of RLX3
(Tanaka et al., 2005) (see Discussion).
RLX3-LI in neurons of the NI
A discrete population of neuronal cell bodies located in
the NI was found to contain RLX3-LI (Fig. 3AE=), con-
sistent with earlier descriptions of high levels of RLX3
mRNA in these cells (Burazin et al., 2002; Liu et al.,
2003b). The NI is a distinct cell group found in the
caudoventral regions of the pontine periventricular gray,
just below the fourth ventricle and based on cytoarchi-
tecture it consists of two distinct subnuclei, the NIc and
NId (Olucha-Bordonau et al., 2003). The NIc is a com-
pact group of multi-polar neurons that lie near the mid-
line, dorsal to the medial longitudinal fascicle. The NId is
a group of more diffusely arranged neurons that extend
laterally from the NIc, along the ventral border of the
dorsal tegmental nucleus (see Olucha-Bordonau et al.,
2003). Staining of coronal sections through the 600
800 m rostrocaudal extent of this nucleus revealed that
the majority of neurons containing dense RLX3-LI were
located in the NIc, while some more scattered positive
cells were found in the NId. In some sections, specifi-
cally stained cells were also found along the medial
edge of the fourth ventricle (see below). Immunoreac-
tivity was strongest in the cytoplasm of neurons and
little or no staining was observed in the nuclei of these
cells. Immunoreactivity was also observed in the proxi-
mal processes of these cells and in fiber networks ad-
jacent to the NI that extended in a lateroventral direction
to the dorsomedial tegmental area and in a laterodorsal
direction, bordering the posterodorsal tegmental nu-
cleus. A few immunoreactive fibers were observed
in the midline below the NI, between the medial longitu-
dinal fasciculi, descending toward the predorsal bundle.
A sparse number of cells in the periaqueductal gray
and an area dorsal to the substantia nigra also con-
tained RLX3-LI (Fig. 3F, G), as reported (Tanaka et al.,
2005).
170 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 3. Distribution of RLX3-LI neurons in rat brain. (AD) Representative photomicrographs through the rostral to caudal extent of the NI, stained with
AS-R385101 antiserum and conventional DAB immunohistochemistry. (E, E=) High resolution images reveal staining in the cytoplasm of cells. (F, G)
Sparse cell staining was also detected in the periaqueductal gray and substantia nigra. Scale bars120 m (AD, F, G), 60 m (E, insets F, G) and
25 m (E=).

Neurochemical nature of neurons in the NI
containing RLX3-LI
In studies aimed at identifying the neurochemical phe-
notype and the nature of the primary transmitter used by
RLX3-containing neurons in the NI, coronal sections
through the NI of colchicine pre-treated rats were used
in double-label immunofluorescence experiments. Using
the AS-R385-101 antiserum, the peripheral somatic cyto-
plasm and proximal dendritic or axonal processes of NI
neurons were stained throughout rostral, intermediate,
and caudal levels of the NI (Fig. 4AC). Sparse immu-
noreactive fibers were also detected within the NI. Using
a monoclonal antibody against GAD65, specific immu-
noreactivity was detected in numerous cell bodies and
fibers within the NI, as described (Olucha-Bordonau et
al., 2003). Notably, GAD65-LI was not present in cells of
the neighboring posterior dorsal tegmental nucleus.
The specificity of the GAD staining was verified using
conventional DAB-chromagen staining of normal brain
sections that displayed strong punctate immunoreactiv-
ity associated with fibers and terminations in the supe-
rior colliculus and substantia nigra (data not shown;
Weiner et al., 2005). Overlaying of duplicate images of
RLX3-LI and GAD65-LI of the NI captured by confocal
microscopy demonstrated that most RLX3-positive
cells were GAD65-positive (Fig. 4AC); and allowing
for the technical constraint of differing degrees of
detection sensitivity for the two antigens, this result
 S. Ma et al. / Neuroscience 144 (2007) 165190 171

Fig. 4. Double label immunofluorescence for RLX3 in the NI. Confocal photomicrographs illustrating (A, B, C) localization of RLX3-LI and (A=, B=, C=)
GAD in neuronal cell bodies of the NI (A, B, C). Merged immunofluorescent images reveal that most, if not all, RLX3-LI cells co-localize with GAD
(white arrows). In some sections, GAD-positive only cells were detected (hollow arrows). Confocal photomicrographs illustrating (D, E, F) localization
of RLX3 and (D=, E=, F=) calbindin (CB) in neuronal cell bodies of the NI. (D, E, F) Merged immunofluorescent images reveal approximately 30%
of cells show co-localization of RLX3-LI with calbindin (white arrows). Numerous RLX3-LI-only and CB-only cells were observed (hollow arrows). Scale
bars30 m (A, A=, A), 85 m (D, D=, D) and 20 m (BC, EF).

suggests that all RLX3 cells co-express GAD and pro-
duce GABA. Conversely, in some sections, occasional
GAD-only immunoreactive cells were also observed
(Fig. 4C=), indicating that not all GABA cells in the area
express RLX3.
In separate studies, a monoclonal calbindin D-28K
antibody strongly stained the cytoplasm of a population
of neuronal soma and nerve fibers throughout the NI, but
no staining was observed in the neighboring posterior
dorsal tegmental nucleus (Fig. 4DF). Thus, a distinct
population of cells contained RLX3- and calbindin-LI,
while some contained RLX3-LI only, and some calbin-
din-LI only (Fig. 4DF). Analysis of at least five sections
from three brains indicated approximately 30% co-local-
ization of RLX3 and calbindin within the NI and verified
the specificity of the Alexa-594 and FITC fluorophores,
revealing little to no cross-fluorescence.
Using a guinea-pig antibody against VGlut2, no immu-
noreactive cell bodies were observed in the NI of colch-
icine-treated rats, but numerous immunostained termina-
tions were observed (data not shown), indicating a sub-
stantial (sub-cortical?) glutamate innervation of the region.
However, further studies are required to determine the
precise nature and extent of glutamate transmission in the
172 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 5. Distribution of RLX3-LI in parasagittal sections of rat brain. (AF) Representative schematics adapted from Swanson (1998, 1999). (A=F=)
Darkfield photomicrographs illustrating the distribution of RLX3-LI. Scale bar70 m.

NI and this aspect was not further explored in the current
study.
Ascending projections positive for RLX3-LI
In line with the primarily single locus of neurons that ex-
press RLX3 mRNA and peptide and anatomical evidence
for broad, discrete projections of NI neurons within the
forebrain, the distribution of RLX3-LI observed by immu-
noperoxidase staining was similarly restricted and the
more prominent RLX3-positive fiber/terminal fields were
observed in areas known to be targets of NI efferents.
Thus, ascending axons, fibers and terminals containing
RLX3-LI were detected throughout the rhinencephalon,
telencephalon, diencephalon and mesencephalon (Figs. 5,
6 and 7; Table 1).
In initial experiments conducted using parasagittal sec-
tions of rat brain, two major fiber pathways were detected,
and smaller branches of these pathways were observed
 S. Ma et al. / Neuroscience 144 (2007) 165190
diverging to innervate nearby structures. Midline sections
revealed a population of RLX3-LI cells in the NI (Fig. 5A,
A=) and their axonal projections to the ventral mesenceph-
alon and hypothalamus (Fig. 5B, B=). A subgroup of these
fibers also terminated at the level of the dorsal raphe and
coursed dorsally to the colliculi (data not shown). The
majority of immunoreactive fibers continued through the
bed nucleus of the stria terminalis adjacent to the anterior
commissure (Fig. 5C, C=) to the septum (Fig. 5D, D=).
Other fibers in the anterior hypothalamic region continued
into the prefrontal cortex along the corpus callosum, inner-
vating the cingulate and retrosplenial cortices (Fig. 5E, E=,
F, F=). A small cluster of fibers was also detected entering
the prefrontal cortex and terminating in the anterior olfac-
tory region and olfactory bulb (data not shown). On the
basis of these findings, a series of brains was processed in
the coronal plane and analyzed in detail (Figs. 6 and 7).
Rhinencephalon
In the olfactory bulb, a small cluster of immunoreactive
elements was observed adjacent to the olfactory ventricle
of the ependyma and subependymal layer (Figs. 6A, A=,
7A, B). Stained fibers were also observed in the nearby
internal granule cell layer. This cluster was observed near
the midline throughout the rostrocaudal extent of the olfac-
tory bulb and olfactory nuclei/cortex (Figs. 6B, B=, 7A, B).
Fibers were also observed coursing through the medial
anterior olfactory nucleus and continuing upwards into the
cingulate cortex, adjacent to the forceps minor of the cor-
pus callosum (Figs. 6C, C=, D, 7C, D).
Telencephalon
A substantial number of ascending RLX3-LI containing
fibers were detected coursing through the hypothalamus
and entering the septal regions, where they provided
dense inputs to the medial (MS) and lateral septum (LS)
and nucleus of the diagonal band (NDB) (Figs. 6EK=,
7DF). In the LS, stained fibers were dense in the inter-
mediate region and moderate to sparse in the ventral and
dorsal regions (Figs. 6F, F=, G, I, 7F). A moderate number
of immunoreactive fibers were present in the anteromedial
and ventral divisions of the bed nucleus of the stria termi-
nalis and septo-hypothalamic nucleus (Fig. 6J) and hori-
zontal and vertical limbs of the NDB (Fig. 6K, K=). Further-
more, due to the high density of immunoreactive fibers in
the medial septum, sections through this area were used in
control experiments to verify the specificity of the proRLX-3
polyclonal antiserum. Sections incubated in antiserum pre-
absorbed with 1 g/ml of rat C-peptide antigen lacked any
specific fiber staining and displayed only non-specific cell
body staining that was also observed with the non-ab-
sorbed antiserum (see Experimental Procedures and Fig.
2 for further controls).
In the cortex, fibers and processes containing RLX3-LI
were observed throughout the rostrocaudal extent of the
cingulum bundle that lies near the midline adjacent to the
cingulum and labeled fibers extended from this bundle to
provide sparse inputs to the anterior cingulate and retro-
splenial cortices. A bundle of fibers was detected immedi-
173
ately lateral to the forceps major of the corpus callosum
and provided a dense innervation of the retrosplenial gran-
ular and agranular cortices (Figs. 6L, L=, 7DN; see also
Fig. 5E=, F=). In addition, a moderate number of immuno-
reactive fibers appeared to terminate in the compact, dor-
sal endopiriform nucleus (Figs. 6M, M=, 7DI).
Diencephalon
At anterior levels of the hypothalamus, dense immunore-
active fibers were observed in the lateral preoptic area
(LPO), while fewer fibers were detected in the medial
preoptic area (MPO) (Figs. 6N, N=, 7F). At rostral levels of
the hippocampus, dense fibers were present in the hip-
pocampal fimbria, septo-fimbrial nucleus and fimbria;
whereas fibers in the triangular septal nucleus were more
sparse (Fig. 6OP). In the intermediate hypothalamus,
dense axons/fibers coursed through the medial forebrain
bundle, lateral to the ventromedial hypothalamic nucleus
and adjacent to the optic tract (Figs. 6Q, 7GI); with a
prominent group traveling medially from the bundle to gen-
erate an apparent terminal field in the dorsomedial hypo-
thalamic nucleus (Fig. 6R). A second fiber group coursed
laterally along the optic tract and sparse fibers were ob-
served in the central and basomedial amygdala nuclei (Fig.
7H). Immunoreactive fibers were also observed in the
posterior hypothalamic nucleus (Fig. 7J).
In the caudal hippocampus, heavily stained fibers were
observed in the alveus of the hippocampus and sparse
fibers were detected within the pyramidal cell layer and
stratum lucidum/radiatum of the CA1-2 fields and fimbria
(Figs. 6SU, 7K, L). A very dense cluster of stained fibers
was observed in the pyramidal cell layer of CA3 and
throughout molecular, granule and polymorphic layers of
the dentate gyrus (Figs. 6U, U=, 7K, L). The ventral
hippocampus and dentate gyrus consistently contained
a noticeably more dense network of immunoreactive
fibers than the dorsal hippocampus. In addition, dense
fibers were detected in the neighboring amygdalo-hip-
pocampal area (Figs. 6U, 7J, K); and in temporal regions
of the entorhinal cortex and pre- and para-subiculum
(Fig. 7M, N).
In the thalamus, a distinct terminal field was observed
in the centromedial nucleus (Figs. 6V, 7GI) and some
fibers also coursed through the anterior paraventricular
thalamic nucleus. A low density of terminations containing
RLX3-LI was also observed in the adjacent centrolateral
thalamic nucleus (Fig. 7H).
Mesencephalon
In parasagittal sections, a bundle of immunoreactive fibers
was observed ascending from cells of the NI caudoven-
trally through the mesencephalon, parallel to the decussa-
tion of the superior cerebellar peduncle, (Fig. 5A=, B=). A
small number of fibers coursed upwards to the superior
colliculus, notably to the optic nerve/superficial gray layer
(Figs. 6W, W=, 7L, M). Coronal sections through the rostral
pons revealed immunoreactive fibers coursing through the
pontine periventricular regions that continue rostrally into
the dorso- and ventro-lateral PAG and the dorsal raphe
174 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 6. Distribution of RLX3-LI in coronal sections of rat brain. (AFf) Darkfield photomicrographs illustrating the distribution of RLX3-LI in fibers and/or
terminals at low resolution and (A=Ee=) high resolution. (E) High resolution images of RLX3-LI in the medial septum. Scale bars60 m (AFf),
25 m (A=Ee=) and 5 m (E).
 S. Ma et al. / Neuroscience 144 (2007) 165190 175

Fig. 6. (Continued).

nucleus (DR) (Figs. 6XY, 7KN). Within the geniculate observed in the intergeniculate leaflet (IGL) and sparse
nuclei, a moderate density of immunoreactive fibers was fibers were present in the adjacent dorsolateral and ven-
176 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 6. (Continued).

trolateral parvocellular geniculate nuclei (Figs. 6Z, Z=, 7J, lary levels of the hypothalamus (Figs. 6Aa, Aa=, 7J). Here,
K). A major deposit of RLX3-LI was observed at mammil- the majority of immunoreactive fibers were observed in the
 S. Ma et al. / Neuroscience 144 (2007) 165190 177

Fig. 6. (Continued).

medial mammillary nucleus, and a sparse number of fibers
were seen in the supra- and lateral-mammillary nuclei. These
fibers continued through the interfascicular nucleus, ventral
tegmental area and medial paranigral nucleus into the inter-
peduncular nucleus, particularly the apical, dorsolateral, and
rostral subnuclei (Figs. 6Bb, Bb=, 7L). Within the rostral pons,
RLX3-LI was detected in cells bodies of the NI and within
proximal processes and fiber networks adjacent to the NI that
extended in a lateroventral direction to the dorsomedial teg-
mental area and in a laterodorsal direction, bordering the
posterodorsal tegmental nucleus (Figs. 6DdDd, 7O). A few
immunoreactive fibers were observed in the midline below
the NI, between the medial longitudinal fasciculi, descending
toward the predorsal bundle (Fig. 6Dd).
Descending projections positive for RLX3-LI
A sparse number of fibers containing RLX3-LI were de-
tected in the metencephalon. Some were observed cours-
ing through the prepositus hypoglossal nucleus (Figs. 6Ee,
Ee=, 7P, Q), but the nucleus of the solitary tract contained
little to no immunoreactive fibers (Fig. 6Ff), despite
GPCR135 mRNA expression (see below). Sparse immu-
noreactive fibers were observed in some sections through
the lateral reticular nucleus (Fig. 7R).
178 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 7. Schematic representation of the distribution of RLX3-LI throughout the rat brain (red lines). Schematics adapted from Swanson (1998, 1999).
For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
 S. Ma et al. / Neuroscience 144 (2007) 165190 179

Fig. 7. (Continued).

Distribution of GPCR135 mRNA in relation to
RLX3-LI
GPCR135 mRNA was widely distributed throughout the rat
brain, as reported (Sutton et al., 2004). The distribution
correlated well with that of specific binding sites for the
selective chimeric peptide, [125I]-R3/I5 (Sutton et al., 2004;
Liu et al., 2005a), and in line with the now substantial
molecular and pharmacological evidence that RLX3 is
the cognate ligand for GPCR135 (Sutton et al., 2004,
2006). In the following section, higher-resolution images
of GPCR135 mRNA are presented and correlated with the
distribution of RLX3-LI.
Rhinencephalon
Moderate to high levels of GPCR135 mRNA were ob-
served in specific cell layers of the olfactory bulb, in par-
180 S. Ma et al. / Neuroscience 144 (2007) 165190

Table 1. Regional distribution of RLX3-LI in rat brain Table 1. continued

Brain region Relative Brain region Relative

abundancea abundancea

Rhinencephalon Posterior hypothalamic n.

Anterior olfactory n., medial Supramammillary n.
Ependyma and subependymal layer/ Ventromedial hypothalamic n.
olfactory ventricle Thalamus
Olfactory bulb Centrolateral n.
Glomerular layer Centromedial n.
Internal granule cell layer Habenular n.
Telencephalon Intergeniculate leaflet
Accumbens n., shell Intermediodorsal thalamic n.
Amygdala Medial geniculate n.
Basomedial n. Paraventricular thalamic n.
Central n. Peripeduncular n.
Medial n. Precommissural n.
Bed n. of the stria terminalis Posterior thalamic area
Lateral division, dorsal part Reuniens thalamic n.
Medial division, anterior part Rhomboid thalamic n.
Ventral division Mesencephalon
Cerebral cortex Caudal linear n. raphe
Entorhinal cortex Central grey
Frontal cortex area 1/2 Dorsal
Infralimbic cortex Medial (Cell bodies)
Laminal dissecans, entorhinal Dorsal raph n.
cortex Edinger-Westphal n.
Medial/ventral orbital cortex Interfascicular n.
Para/presubiculum Interpeduncular n.
Retrosplenial agranular cortex Caudal subn. /
Retrosplenial granular cortex Dorsolateral subn.
Claustrum Intermediate subn.
Dorsal endopiriform n. Lateral subn.
Hippocampal formation Rostral subn.
Alveus Paranigral n.
Amygdalohippocampal area Pontine n.
CA1 field Rostral linear n. raphe
CA2 field Superior colliculus
CA3 field Optic nerve layer
Dentate gyrus Superficial gray layer
Fimbria Ventral tegmental area
Fornix Substantia nigra, compacta (Cell bodies)
Nucleus diagonal band Rhombencephalon
Horizontal limb Dorsomedial tegmental area
Vertical limb Lateral reticular n. /
Septum Locus coeruleus
Lateral septal n., dorsal / Median raphe
Lateral septal n., intermediate Posterodorsal tegmental n.
Lateral septal n., ventral Prepositus hypoglossal n.
Medial septal n. Raphe pontis n.
Septofimbrial n. Solitary tract n. /
Septohypothalamic n. Nucleus incertus
Diencephalon Nucleus incertus, compacta (Cell bodies)
Hypothalamus Nucleus incertus, dissipata (Cell bodies)
Anterior hypothalamic n.
Arcuate hypothalamic n. a
Relative abundance RLX3-LI: , not detectable; , weak; , low;
Dorsomedial hypothalamic n. , moderate; , high; , intense.
Medial mammillary n.
Medial preoptic area ticular the internal plexiform layer with much lower levels in
Lateral hypothalamic area/medial the internal granular and glomerular layers (Fig. 8A). Low
forebrain bundle
Lateral preoptic area
to moderate levels of immunoreactive terminations were
Paraventricular hypothalamic n. observed in the olfactory bulb adjacent to the olfactory
Periventricular hypothalamic n. ventricle of the ependyma and subependymal layer and
nearby internal granule cell layer (Fig. 6A, A=). In some
 S. Ma et al. / Neuroscience 144 (2007) 165190
sections, sparse fibers were observed in the internal plex-
iform layer where GPCR135 mRNA is most abundant. In
the anterior olfactory nuclei, scattered cells expressing
GPCR135 were observed in the various subnuclei and
throughout the prefrontal cortex (Fig. 8B, B=). A tract of
RLX3-LI was observed adjacent to the ependyma and
subependymal layer/olfactory ventricle at the level of the
anterior olfactory nucleus (Fig. 6B, B=). Moderate levels of
RLX3-LI fibers were also present coursing from the medial
anterior olfactory nucleus through the medial and ventral
orbital cortex and cingulate cortex, adjacent to the forceps
minor corpus callosum (Fig. 6CD). Only sparse fibers
were observed in the lateral frontal and parietal cortices,
associated with prominent GPCR135 mRNA expression in
these areas.
Telencephalon
Moderate to high levels of GPCR135 mRNA were ob-
served in parts of the MS and LS (Fig. 8CE=), regions that
contain the highest density of RLX3-LI (Fig. 6EI). Higher
resolution analysis of autoradiograms revealed that the
ventral medial septum and the adjacent NDB (dorsal re-
gions) contained low GPCR135 expression, while moder-
ate levels were detected in the ventral border areas of the
diagonal band (Fig. 7D=). The central medial septum/diag-
onal band area contains abundant RLX3-LI (Fig. 6E, K,
K=), suggesting that much of this immunoreactivity may
represent peptide being trafficked in axons to nerve termi-
nals in the dorsomedial septum and/or LS. Within the LS,
GPCR135 mRNA appears restricted to the intermediate
zone and is relative scarce in the dorsal subnucleus (Fig.
8CE=). This is mirrored by the distribution of RLX3-LI in
fibers, which are enriched in the intermediate not dorsal
subnucleus of LS (Fig. 6G, I).
In the hippocampus, relatively few GPCR135-positive
cells were observed in the dorsal CA1-3 fields (Fig. 8H,
H=), whereas GPCR135-positive neurons were more abun-
dant in the subgranular layer of dentate gyrus particularly
in the caudal (ventral) zones (Fig. 8I, I=). This is mirrored by
levels of RLX3-LI, which are high in fibers of the dentate
gyrus subgranular and polymorphic zones of the ventral
hippocampus (Fig. 6U, U=) and lower in the dorsal CA1-3
areas (Fig. 6S, S=).
Diencephalon
In the anterior hypothalamus, moderate to high densities of
GPCR135 mRNA-positive neurons were evenly distributed
across the MPO and LPO (Fig. 8E, F), whereas significant
axonal profiles strongly stained for RLX3-LI were more
confined to the LPO and lower levels of RLX3-LI were
observed in the medial region (Fig. 6N, N=). High levels of
GPCR135 mRNA were also observed in the dorsolateral
bed nucleus of the stria terminalis and septo-hypothalamic
nucleus (Fig. 8E, E=), and these regions receive a relatively
modest innervation of elements containing RLX3-LI (Fig.
6J). Moderate to high levels of GPCR135 mRNA were
observed in lateral hypothalamus (LH) (Fig. 8F), with cor-
responding high levels of RLX3-LI in axons/fibers in the
medial forebrain bundle (Fig. 6QQ). GPCR135 mRNA
181
was also present in the paraventricular and dorsomedial
hypothalamic nuclei (Fig. 8G, H), which contain moderate
levels of RLX3-LI (Fig. 6R).
In the thalamus, GPCR135 mRNA was concentrated in
neurons of the centromedial, centrolateral and paraven-
tricular nuclei (PVN) (Fig. 8FH), complementary to mod-
erate levels of RLX3-LI fibers in these areas (Fig. 6V). High
levels of GPCR135-expressing cells were also observed in
the central and basomedial amygdala nuclei (Fig. 8G, G=
H), but only sparse RLX3-LI was observed in these areas
(Fig. 5; Table 1).
Mesencephalon
Moderate levels of GPCR135 mRNA were observed in the
periaqueductal gray (Fig. 8J, K), which also contained
moderate accumulations of RLX3-LI fibers (Fig. 6X, X=). A
small number of RLX3-LI fibers appeared to course up-
wards to the superior colliculus, notably the optic nerve/
superficial gray layer (Fig. 6W, W=) that also contains
moderate to high levels of GPCR135 mRNA (Fig. 8I, I).
The nearby IGL contained moderate levels of both
GPCR135 mRNA and RLX3-LI (Fig. 8I, I and 6Z, Z=) and
sparse fibers were present in the adjacent dorsolateral and
ventrolateral parvocellular geniculate nuclei (Fig. 6Z). An
abundance of RLX3-LI fibers was observed in the interpe-
duncular nucleus, particularly in the apical, dorsolateral,
and rostral subnuclei (Fig. 6Bb, Bb=), whereas GPCR135
mRNA appeared restricted to, or at least highly enriched
in, the lateral subnuclei (Fig. 8J, J=).
In the hindbrain, dense GPCR135 mRNA levels were
detected in the NIc and NId (Fig. 8L, L=), where the majority
of RLX3-containing neurons in the brain are located (Figs.
3 and 4). Low levels of GPCR135 expression were also
observed in the prepositus hypoglossal nucleus (Fig. 8M),
which contains a similarly sparse density of RLX3-LI (Fig.
6Ee, Ee=). Low to moderate levels of GPCR135 mRNA
were detected in the nucleus of the solitary tract and spinal
trigeminal tract nucleus (Fig. 8N, N=), but little to no
RLX3-LI was detected in the current material (Fig. 6Ff;
data not shown).
GPCR135 mRNA and [125]-R3/I5 binding sites
As reported, the regional distribution of cells expressing
GPCR135 mRNA and specific binding sites for the
GPCR135-selective chimeric peptide, [125I]-R3/I5, gener-
ally correlate well in rat brain (Sutton et al., 2004; Liu et al.,
2005a), but notably in some areas there is differential
between the relative proportion and juxtaposition of mRNA
and binding. For example, the neocortex contains moder-
ate to high levels of GPCR135 mRNA expression in cells
evenly distributed across the outer layers, whereas [125I]-
R3/I5 binding sites are relatively low compared with some
limbic and subcortical areas and appear more evenly dis-
tributed in specific cortical layers (Fig. 9A, A=). In these
same sections, little or no binding of [125I]-R3/I5 was ob-
served in the LS, bed nucleus of the stria terminalis and
preoptic areas of the hypothalamus, areas that contain
abundant GPCR135 mRNA (Fig. 9B, B=). These observed
differences do not appear to be due to any major technical
182 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 8. Distribution of GPCR135 mRNA in coronal sections of rat brain. Representative autoradiographs at (BN) low resolution and (A, B=O=) high
resolution. Scale bars675 m (A, B=), 1.5 mm (CK), 400 m (C=O=) and 2 mm (LO).
 S. Ma et al. / Neuroscience 144 (2007) 165190 183

Fig. 8. (Continued).

limitations of either assay, as for example the superior and binding sites (Fig. 9B, B=), indicating that the radioli-
colliculus displays high levels of both GPCR135 mRNA gand is able to bind to GPCR135 when present; although
184 S. Ma et al. / Neuroscience 144 (2007) 165190

Fig. 9. Comparison between representative autoradiographs of (A, B) GPCR135 mRNA and (A=, B=) [125I]-R3/I5 binding sites. Areas of the
retrosplenial and cingulate cortices consistently showed relatively sparse GPCR135 mRNA and [125I]-R3/I5 binding sites in comparison to the rest of
the cortex (black arrows). Similarly high levels of both GPCR135 mRNA and [125I]-R3/I5 binding sites were detected in the superior colliculus (white
arrows). Non-specific binding was assessed in the presence of 100 nM RLX3 in the incubation buffer (A, B). Scale bar2 mm.

notably in this region the receptor protein reflected by
specific binding sites seems located in an adjacent collicu-
lar layer, perhaps associated with the corresponding den-
dritic fields of the GPCR135 expressing cells.
DISCUSSION
where this strict correlation of RLX3 innervation and
GPCR135 density was not so clearly observed. This is
discussed in further detail below.
RLX3-LI and other signaling systems in neurons of
the NI

These studies have identified the extensive, topographical
distribution of RLX3-LI throughout the adult, male rat brain,
with expression detected in specific regions/nuclei of the
olfactory bulb, neocortex, limbic forebrain, hypothalamus,
thalamus, midbrain, pons and medulla. Highest densities
of nerve fibers and terminals were observed in the medial
septum, LPO, LH/medial forebrain bundle and ventral hip-
pocampus; and additional fibers were observed in olfactory
bulb and olfactory and frontal/cingulate cortices, bed nu-
cleus of the stria terminalis, dorsal endopiriform, inter-
geniculate, and supramammillary nuclei, and the periaq-
ueductal gray and dorsal raphe. All these areas generally
contained a correspondingly abundant and regionally re-
stricted level of GPCR135 mRNA. These data along with
earlier findings (Burazin et al., 2002; Tanaka et al., 2005)
suggest that RLX3 is produced in high concentrations in
neuronal soma of the NI, and is transported axonally and
released at presynaptic sites throughout the forebrain onto
postsynaptic GPCR135 (receptors). GPCR135 is also po-
sitioned to act as an autoreceptor for locally produced
RLX3 in the NI. In the material examined, the cortex,
olfactory bulb, and nucleus of the solitary tract were areas
In agreement with previous immunohistochemical studies
using a rabbit polyclonal antibody (R6) that recognizes
RLX3 as well as RLX (Burazin et al., 2002) and a mono-
clonal antibody raised against the N-terminus of the RLX3
A-chain (Tanaka et al., 2005), the current study demon-
strated abundant RLX3-LI in neurons of the rat NI and
demonstrated that most, if not all, RLX3 neurons coex-
press GAD65, indicative of GABAergic neurons. Further-
more, a subpopulation of these RLX3-GABAergic neurons
also contains the calcium binding protein, calbindin.
Tanaka et al. (2005) recently demonstrated that almost all
RLX3 neurons co-express CRF-R1, but less than 3% of
RLX3 neurons in the NI demonstrated 5-HT-LI. In addition,
65% of RLX3-positive neurons display elevated nuclear
Fos levels, after intracerebroventricular injection of 1 g
CRF, suggesting that there is a functional interaction be-
tween these two signaling systems (see below).
Other transmitters, peptides or relevant marker pro-
teins that appear to be present in the rat NI include 5-HT
(Sutin and Jacobowitz, 1988; Olucha-Bordonau et al.,
2003; Tanaka et al., 2005), cholecystokinin (Cho et al.,
1983; Sutin and Jacobowitz, 1988; Olucha-Bordonau et
 S. Ma et al. / Neuroscience 144 (2007) 165190
al., 2003), acetyl cholinesterase (Sutin and Jacobowitz,
1988; Olucha-Bordonau et al., 2003), a potential bomb-
esin-like peptide, recognized by a ranatensin antisera
(Chronwall et al., 1985), substance P, galanin (Sutin and
Jacobowitz, 1988) and neurotensin (Jennes et al., 1982;
Sutin and Jacobowitz, 1988), although several early map-
ping papers referred to this nucleus as a ventromedial area
adjacent to the laterodorsal tegmental nucleus or the ven-
tromedial pontine central gray. Further studies will be re-
quired to determine the existence of any subpopulations of
NI neurons with particular neurochemical profiles and pos-
sibly distinct functional roles in the rat and in other species
including human.
Connections of the NI
The anatomical connectivity of the NI has been well char-
acterized in the rat (Goto et al., 2001; Olucha-Bordonau et
al., 2003) following renewed interest in its function stem-
ming from the high density of CRF-R1 observed in this
nucleus (Potter et al., 1994; Bittencourt and Sawchenko,
2000). In fact, due to its anatomical proximity to the fourth
ventricle, it was postulated to be a site at which any circu-
lating, endogenous CRF produced by several possible
sources including neurons in the paraventricular hypotha-
lamic nucleus and central amygdala (Sawchenko and
Swanson, 1985), might elicit some central effects related
to stress behavior (Bittencourt and Sawchenko, 2000).
Retrograde tracing experiments have identified that the
primary inputs to the NI arise from neurons of the prefrontal
cortex, septum-diagonal band, lateral and posterior hypo-
thalamus, habenula, dorsal raphe, and supramammillary
and interpeduncular nuclei (Goto et al., 2001; Olucha-
Bordonau et al., 2003). This strongly suggests that the NI
is in a position to integrate information relating to behav-
ioral planning and responses.
The current immunohistochemical experiments using a
polyclonal antiserum against a portion of the proRLX3
C-peptide that is highly homologous in human, rat, and
mouse (Bathgate et al., 2002), revealed a topographic
distribution of RLX3-LI positive neural projections that very
closely matches that of efferent projections of the NI
mapped using anterograde tract-tracing methods (Goto et
al., 2001; Olucha-Bordonau et al., 2003). Adjacent to the
NI proper, RLX3-LI fibers were found diverging laterally
and throughout the more rostral mid- and fore-brain, with
immunoreactive projections reaching various hypotha-
lamic, thalamic, limbic, and cortical target areas. Consis-
tent with observations in NI efferent projection studies,
immunoreactive fibers from this nucleus appear to join the
medial forebrain bundle (Olucha-Bordonau et al., 2003),
the major fiber tract connecting the fore- and mid-brain with
the hindbrain. In some regions, RLX3-LI fibers appear to
represent peptide en transit, for example the hippocampal
fimbria contains low levels of GPCR135 mRNA, but abun-
dant RLX3-LI fibers.
During the course of the current studies, Tanaka et al.
(2005) described a very similar map of RLX3-containing
fibers throughout the rat brain using a RLX3 A-chain-
directed monoclonal antibody and demonstrated that
185
RLX3 immunoreactivity was localized in dense-core vesi-
cles in neuronal perikarya and synaptic terminals, consis-
tent with the potential release of RLX3 from neurons fol-
lowing depolarization. A majority (80%) of RLX3 neurons
co-expressed CRF-R1 in the rat NI and a high proportion of
these cells were responsive to i.c.v. administration of CRF
and prolonged water immersion-restraint stress (2 6 h) in
rats (Tanaka et al., 2005). Studies in our laboratory have
also investigated a possible stress-related role of RLX3
and interaction with CRF in rats undergoing forced swim-
ming and observed an increase in RLX3 mRNA within
30 60 min after a 10-min forced swim (Banerjee et al.,
2005). Together these findings support a role for RLX3 in
the stress response that occurs following activation of the
CRF system (see below).
Comparative analysis of the distribution of RLX3-LI
described in the current study and that of Tanaka et al.
(2005) and anterogradely labeled fiber tracts from the NI
(Goto et al., 2001; Olucha-Bordonau et al., 2003) revealed
a generally good correlation. However, minor discrepan-
cies were noted, such as the presence of low levels of
RLX3-LI observed in the olfactory bulb/cortices in the cur-
rent study, which were not reported elsewhere (Goto et al.,
2001; Olucha-Bordonau et al., 2003; Tanaka et al., 2005).
Furthermore, dense anterogradely labeled fibers were ob-
served descending to the medial accessory inferior olive
(Goto et al., 2001), but were not observed in the current
study (see also Tanaka et al., 2005).
The polyclonal antibody used was raised against a
conserved region of the RLX3 C-peptide, and therefore
may strictly be indicative of the presence of a prepro- or
pro-form of RLX3. However, given the nature of the find-
ings it appears likely that the C-peptide is axonally-trans-
ported and that further enzymatic processing occurs away
from the neuronal cell body probably within the dense core
vesicles during axonal transport and/or at the final desti-
nation in the nerve terminal bouton. Currently nothing is
known of the function of the RLX3 C-peptide, but it is
possible that this peptide may also be bioactive. This is the
case for the precursor of ACTH and -endorphin, which
contains more than one active peptide in its coding se-
quence (Nakanishi et al., 1979). To date however, there is
no information on the metabolic fate of relaxin C-peptides.
The structurally-related insulin peptide also generates a
C-peptide fragment during its biosynthesis, from proinsulin
to mature insulin, and C-peptide-LI has been detected in
the cytoplasm of the soma and the proximal part of apical
dendrites of some pyramidal cells in the human neocortex
and hippocampus (Dorn et al., 1982). In studies in rat, the
insulin C-peptide has been implicated as a neuronal anti-
apoptotic agent and deficiency of insulin C-peptide might
have a role in some CNS impairments that follow diabetes
encephalopathy (Li and Sima, 2004). There have been no
investigations to date of the specific steps of RLX3 biosyn-
thesis and degradation in neurons.
Distribution of GPCR135 mRNA
A comparison of the distribution of RLX3-LI nerve fibers
and terminals with that of GPCR135 mRNA and [125I]-
186 S. Ma et al. / Neuroscience 144 (2007) 165190
R3/I5 binding sites revealed a good correlation in most
brain regions. However, some mismatches between the
relative densities of innervating fibers and postsynaptic
receptors were observed, particularly in the neocortex
where GPCR135 is distributed throughout the different
regions of the neocortex particularly in the outer layers,
but RLX3-LI fibers were only consistently observed in
midline areas, such as the frontal, cingulate and retro-
splenial cortices. Another apparent discrepancy was ob-
served in the nucleus of the solitary tract that contains
moderate levels of GPCR135 but little to no evidence of
RLX3 innervation. In this regard, mismatches between
the level of innervation by neuropeptide containing pro-
jections and the local density of receptor binding sites
are not uncommon observations (Herkenham, 1987)
and have been noted for the central tachykinin (Beauj-
ouan et al., 2004), galanin (Jacobowitz et al., 2004), and
calcitonin gene-related peptide systems (Morara et al.,
2000). The absence of immunoreactive fibers in regions
of dense receptor gene expression and binding sites
may be a reflection of a high turnover of peptide and
lower levels present in more active nerve terminals that
are more difficult to capture for experimental visualiza-
tion by paraformaldehyde-fixation. This may therefore
be a reflection of high basal activity of RLX3-producing
neurons in these receptor-rich regions.
This may indicate that in the cortex GPCR135 pro-
tein is expressed preferentially in particular parts of the
expressing cells, which by their characteristic pattern
appear to represent a population of GABA interneurons.
Interestingly, dense RLX3-LI fibers are only detected in
the cingulate and retrosplenial cortices indicating that
GPCR135, and possibly its gene expression, may be
regulated by endogenous RLX3. In these same sec-
tions, no to little binding of [125I]-R3/I5 was observed in
the LS, bed nucleus of the stria terminalis, and preoptic
areas of the hypothalamus, areas that are abundant in
GPCR135 mRNA. These observed differences are not
due to major technical limitations of either assay, as the
superior colliculus displays high levels of both GPCR135
mRNA and binding sites, indicating that the radioligand
is fully capable of binding to GPCR135 when present,
and in this region, receptor protein seems located on
dendritic fields of adjacent collicular neurons. However
as RLX3 is a highly hydrophobic peptide and [125I]-R3/I5
autoradiography is conducted with very low concentra-
tion of radioligand (only 7 pM) (Sutton et al., 2004),
some of the differences in mRNA and binding density/
distribution may be due to endogenous RLX3 remaining
tightly bound to GPCR135 in the tissue and preventing
full access of the exogenous radioligand to the receptor
sites. In addition, low levels of binding sites were ob-
served in areas where GPCR135 mRNA was not readily
observed, such as the caudate putamen (see Results Fig. 9).
This suggests that, in some regions, GPCR135 may be
trafficked to axons or presynaptic terminals that are some
distance from the soma.
Putative involvement of RLX3 systems in
stress-related neural circuits
The distribution of RLX3-LI and GPCR135 in multiple ar-
eas of the brain suggests an interaction with circuits/sys-
tems that regulate locomotor activity, arousal, cognition
and stress responses, as originally proposed (Goto et al.,
2001; Olucha-Bordonau et al., 2003).
Septohippocampal-related systems
The septum situated in the medial basal forebrain provides
a major input pathway from its medial nucleus to the hip-
pocampus. Dense RLX3-LI fibers and terminals were de-
tected in the MS and LS nuclei and peripheral parts of both
horizontal and vertical limbs of the NDB, largely consistent
with NI efferent projection patterns. Furthermore, corre-
sponding high densities of GPCR135 mRNA and R3/I5-
binding sites were detected in the septal nuclei. It is known
that fibers arising from the MS and NDB ascend through
the fimbria, dorsal fornix, and supracallosal stria to termi-
nate in all fields of the hippocampal formation, and prom-
inently in the dentate gyrus (Daitz and Powell, 1954; Swan-
son and Cowan, 1977), and the primary cholinergic inner-
vation of the hippocampal formation arises from the MS
and NDB (Amaral and Kurz, 1985). The MS and NDB are
closely associated with the hippocampus, both anatomi-
cally and functionally, and are involved in processes relat-
ing to attentional levels, sleepwakefulness, and anxiety.
Cholinergic and GABAergic neurons of the MS and NDB
are involved in the generation of hippocampal theta rhythm
(Henderson et al., 2004); and a recent study by Nunez et
al. (2006)4 in urethane-anesthetized rats demonstrated
that stimulation of the NI evokes hippocampal theta rhythm
activity, whereas lesions or pharmacological blockade of
the NI abolished theta rhythms evoked by stimulation of
the reticular pons. These data suggest that NI neurons,
including those that produce RLX3, act to relay information
between brainstem structures and the MSNDB in the
control of hippocampal theta rhythm generation.
Furthermore, the septum appears to have a modula-
tory role in mnemonic processing of the hippocampus,
since lesion studies indicate that the septum is not neces-
sary for spatial memory formation (Cahill and Baxter, 2001;
Pang et al., 2001; Vuckovich et al., 2004). Like the baso-
lateral amygdala, the septum may also be involved in the
emotional processes associated with memory information.
Hippocampal cholinergic and septal GABAergic systems
have been reported to act synergistically in modulation of
anxiety in rats (Degroot et al., 2001; Degroot and Treit,
2003). These data and more of a similar nature in the
existing literature on these important brain regions strongly
suggest an involvement of RLX3 and GPCR135 in septal-
and hippocampal-related behaviors that warrants further
investigation.
Hypothalamic systems
Various hypothalamic nuclei displayed dense RLX3-LI, in-
cluding the LPO/MPO, arcuate nucleus, anterior hypotha-
lamic area, LH, PVN, dorsomedial nucleus, pre- and su-
 S. Ma et al. / Neuroscience 144 (2007) 165190
pramammillary nuclei, and posterior hypothalamic nu-
cleus. Of these nuclei, the highest density of fibers/
terminals was observed in the LPO and LH. The LPO is
composed of scattered cells with various morphological
characteristics. The medial forebrain bundle tract is known
to occupy part of the LPO and the RLX3-LI observed
appeared as a combination of axonal profiles within this
tract and local terminations. Previous studies have re-
ported that neuronal activity in the MPO and neighboring
ventrolateral preoptic area promotes sleep. During sleep in
rats, an increase in the number of Fos immunoreactive
neurons has been observed in both rostral and caudal
parts of the MPO suggesting that the preoptic hypotha-
lamic area is involved in promoting sleep processes (Gong
et al., 2000). In monkeys, neurons of the MPO have been
reported to be thermo-sensitive and have an involvement
in the control of thermoregulatory cooling behavior (Hori et
al., 1987). Interestingly, large excitotoxic lesions of the
MPO and LPO eliminated all copulatory behavior in male
rats, and these rats also demonstrated reduced preference
for receptive over non-receptive females (Edwards et al.,
1996). The role for this area and other hypothalamic areas
in arousal and wakefulness, thermoregulation and repro-
duction suggests a possible involvement of RLX3
GPCR135 signaling in these functions, particularly in var-
ious autonomic and neuroendocrine components of be-
havior.
Thalamicmidbrain systems
Early studies report that the peripeduncular nucleus (PP)
of the lateral midbrain has a strong influence on reproduc-
tive behavior in rats, and the current study demonstrated
that the PP receives moderate RLX3-positive fibers. The
PP is interconnected with the auditory, motor, and limbic
systems, which strongly suggests this area might play a
role in female sexual receptivity (as expressed by lordosis
or ultrasound production) or male mounting behavior (Ar-
nault and Roger, 1987). Electrolytic or ibotenate lesions of
the PP significantly reduced the increase in food intake
and aggression to male intruders that is usually elevated in
lactating mothers (Hansen and Kohler, 1984; Hansen and
Ferreira, 1986). These rats also displayed little sexual
receptivity and proceptivity in response to estrogen and
progestin treatment, and the milk ejection reflex appeared
nonfunctional following lesions (Hansen and Kohler, 1984;
Factor et al., 1993). Lesioned male rats failed to ejaculate
on most postoperative observations, though they contin-
ued to mount estrous females (Hansen and Kohler, 1984).
Together with strong connections to other behavioral net-
works, the RLX3GPCR135 system may have a significant
role in male and female reproductive behavior.
Adjacent to the PP is the IGL, which is part of the
circadian visual system in the suprachiasmatic nucleus
and contains a moderate density of RLX3-LI. It is believed
that the IGL integrates information relating to photic and
non-photic visual stimuli and vestibular systems, and its
efferents provide the major NPY and GABA input to the
suprachiasmatic nucleus, via the geniculohypothalamic
tract (Lall and Biello, 2003; Horowitz et al., 2004). Neuro-
187
chemical lesions of the IGL alter both the pattern of circa-
dian entrainment and photoperiodic responsiveness of
hamsters to simulated natural photoperiod (Freeman et al.,
2004; Muscat and Morin, 2006). Recent studies in our
laboratory have revealed cyclical oscillations in RLX3
mRNA in the NI across the 12-h light/dark period (Banerjee
A, Shen P-J, Gundlach AL, unpublished observations),
suggesting a possible interaction of RLX3 signaling and
circadian rhythms, via effects on the IGLsuprachiasmatic
nucleus projections.
Amygdala systems
The current study and that by Tanaka et al. (2005), and
anterograde tract-tracing studies (Goto et al., 2001;
Olucha-Bordonau et al., 2003) observed low levels of
RLX3-LI and NI efferents, respectively in the central, me-
dial, and basolateral amygdala nuclei, despite high levels
of GPCR135 mRNA in the central and basomedial amyg-
daloid nuclei. The role of the amygdala complex in behav-
ior is well studied, particularly its involvement in stress- and
fear-related learning and memory (McGaugh, 2002). The
central amygdala in particular has strong connections with
autonomic output pathways and is involved in modulating
autonomic and parasympathetic activity in response to
stress (Roozendaal et al., 1997; McGaugh et al., 2002;
Everitt et al., 2003), notably the bradycardia and freezing/
immobility observed in rats conditioned to an inescapable
footshock (Roozendaal et al., 1991). Therefore the RLX3
GPCR135 system is implicated in the function of these
amygdala nuclei, which may parallel coincident hypotha-
lamic functions to regulate networks involved in autonomic
activity.
Dorsal raphe systems
In the rhombencephalon of the rat, moderate levels of
RLX3-LI fibers and GPCR135 mRNA were detected in the
DR, the largest raphe nucleus, located in the rostral part of
the dorsal tegmental area and bordered by the Edinger-
Westphal nucleus rostrally and by the NI caudally. The DR
is a major source of serotonergic innervation to the rat
forebrain, particularly in relation to actions of 5-HT in stress
circuits (Lowry, 2002). Efferent connections of the DR
include regions involved in central autonomic control of
stress responses such as the central amygdala and the
PVN (Lowry, 2002) and notably, the DR contains CRF-
immunoreactive fibers, which mediate complex, regionally-
selective excitatory and inhibitory effects on DR activity
(Valentino et al., 2001). Behavioral studies also strongly
indicate a function of the DR in stress-related neural cir-
cuits. In vivo injection and lesion studies in rats (Maier et
al., 1993), reveal that modulation of various neurotransmit-
ter/hormonal systems in the DR, such as 5-HT (Maswood
et al., 1998; Greenwood et al., 2003), opioids (Grahn et al.,
1999), benzodiazepines (Maier et al., 1994, 1995), and
CRF (Hammack et al., 2002), alter behavioral measures of
anxiety and fear 24 48 h after exposure to inescapable
stress in rats, such as tail shock and shuttle box escape
learning paradigms. Furthermore, paradigms associated
with increased anxiety or fear (Beck and Fibiger, 1995;
188 S. Ma et al. / Neuroscience 144 (2007) 165190
Martinez et al., 1998), opiate withdrawal (Chahl et al.,
1996), and infusion of CRF or anxiogenic peptides (Matta
et al., 1997; Bittencourt and Sawchenko, 2000), increased
c-fos gene expression in the DR. This key nucleus appears
to be another important target for RLX3 signaling and
possible modulation of stress- and arousal-related circuits,
via effects on serotonergic activity. It will be important
therefore, to assess whether RLX3-containing terminals
form synapses with 5-HT cells and/or that these 5-HT cells
express GPCR135; and to determine the effects of RLX3
on the activity of these neurons.
CONCLUSIONS
The current study provides a clearer understanding of the
anatomy of the novel RLX3GPCR135 signaling system,
and provides an excellent foundation for experiments
aimed at discovering further details of the neurochemical
anatomy of GPCR135-responsive neurons and circuits;
and efforts to determine the physiological role(s) of this
system in mammalian brain. In this regard, the broad, but
distinct neuroanatomical distribution of this system strongly
suggests an involvement in behaviors such as arousal,
learning and memory, stress, and locomotor activity and
together with preliminary functional evidence suggests that
RLX3GPCR135 signaling has broad modulatory effects
on networks of putative GABA neurons, via co-release with
GABA in key brain areas and important interactions with
circuits containing CRF-like peptides and their receptors,
key players in the autonomic and behavioral changes that
occur in response to stress.
AcknowledgmentsThis research was supported by grants
(983001, 277609 and 300012) from the National Health and Med-
ical Research Council (NHMRC) of Australia, and a collaborative
research agreement between the Howard Florey Institute and
Johnson & Johnson Pharmaceutical Research & Development,
LLC. During these studies S.M. was the recipient of an NHMRC
(Australia) Dora Lush Biomedical Postgraduate Scholarship. The
authors wish to thank Kelli Johnson for technical assistance with
preliminary experiments, Dr. Heather Young for assistance with
confocal microscopy, Diane Nepomuceno for assistance with binding
autoradiography; and formally and gratefully acknowledge the
supply of the anti-GAD monoclonal antibody developed by D. I.
Gottlieb and obtained from the Developmental Studies Hybridoma
Bank, developed under the auspices of the NICHD and main-
tained by the University of Iowa, Department of Biological Sci-
ences, Iowa City, IA, USA. We also thank Brain Foundation Aus-
tralia, the Rebecca L. Cooper Medical Research Foundation and
BAS Medical, San Mateo, CA, USA for their support.
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