Relaxin-3 systems in the brain—The first 10 years

Journal of Chemical Neuroanatomy 42 (2011) 262–275

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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu

Review

Relaxin-3 systems in the brain—The first 10 years Craig M. Smith a,1, Philip J. Ryan a,b,1, Ihaia T. Hosken a,b, Sherie Ma a,c, Andrew L. Gundlach a,d,* a

Florey Neuroscience Institutes, The University of Melbourne, Victoria, Australia Centre for Neuroscience, The University of Melbourne, Victoria, Australia c Department of Medicine, Austin Health, The University of Melbourne, Victoria, Australia d Department of Anatomy and Cell Biology, The University of Melbourne, Victoria, Australia b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 January 2011 Received in revised form 24 May 2011 Accepted 29 May 2011 Available online 14 June 2011

The relaxin-3 gene was identified in 2001 by searching the human genome database for homologues of the relaxin hormone, and was subsequently discovered to encode a highly conserved neuropeptide in mammals and lower species. In the decade since its discovery there have been significant advances in our knowledge of the peptide, including the identification of its cognate receptor (a type 1 G-protein coupled receptor, GPCR135 or RXFP3), an understanding of its structure–activity and associated cellular signalling, and the elucidation of key neuroanatomical aspects of relaxin-3/RXFP3 networks in mammalian brain. The latter studies revealed that relaxin-3 is expressed within GABA neurons of the brainstem including an area known as the nucleus incertus, and that ascending relaxin-3 projections innervate a broad range of RXFP3-rich forebrain areas. These maps provided a foundation for pharmacological and physiological studies to elucidate the neurobiological nature of relaxin-3/RXFP3 signalling in vivo. Recent findings from our laboratory and others suggest the relaxin-3 neural network represents a newly identified ascending arousal system, able to modulate a range of interrelated functions including responses to stress, spatial and emotional memory, feeding and metabolism, motivation and reward, and circadian rhythm and sleep/wake states. More research is now required to discover further important facts about relaxin-3 neurons, such as their various regulatory inputs, and to characterise populations of RXFP3-positive neurons and determine their influence on particular neural circuits, physiology and complex behaviour. ß 2011 Elsevier B.V. All rights reserved.

Keywords: Relaxin-3/insulin-like peptide 7 RXFP3/GPCR135 Neuropeptide modulator Arousal Homeostasis Stress

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery and characteristics of the neuropeptide, relaxin-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution of relaxin-3 neurons in the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurochemistry of relaxin-3 neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The relaxin-3 receptor, RXFP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Distribution of RXFP3 expressing neurons within the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Developmental expression of relaxin-3 and RXFP3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacological, genetic and viral tools for investigating relaxin-3 function . . . . . . . . . . . . . . . . . . . . . . . Putative functional roles of relaxin-3/RXFP3 signalling based on anatomical and experimental evidence . Relaxin-3 neurons: an ascending arousal system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.1. 9.2. Hippocampal theta rhythm-dependent behaviours. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Response to stress. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3. Feeding and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4. Control of sleep/wake states and circadian rhythm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5. Motivation and reward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.6. Other modulatory roles in reproduction and metabolism? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.7. Relevance of relaxin-3 to human disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia. Tel.: +61 3 8344 7324; fax: +61 3 9348 1707. E-mail address: andrew.gundlach@florey.edu.au (A.L. Gundlach). 1 These authors contributed equally to this paper. 0891-0618/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2011.05.013

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11. 12.

Future directions . . Conclusions . . . . . . Acknowledgements References . . . . . . .

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1. Introduction Many neuropeptides modulate essential homeostatic functions, including responses to stress, the sleep-wake cycle, and feeding and appetite. Many of these powerful neural modulators are disturbed in psychiatric disorders, such as anxiety and depression, and due to their broad modulatory roles, neuropeptides have been suggested as excellent putative targets for the pharmacological treatment of a range of disorders (Hokfelt et al., 2003; Insel, 2009). Indeed there is a major ongoing, global scientific endeavour to investigate the role of neuropeptides and their receptors within their underlying neural circuits, in a bid to improve our understanding of – and ultimately treat – such psychiatric disorders (Insel, 2009; Haber and Rauch, 2010). An interesting example is the recently discovered neuropeptide, relaxin-3, which appears capable of modulating essential functions such as responses to stress (Tanaka et al., 2005; Banerjee et al., 2010; Watanabe et al., 2010), arousal (Smith et al., 2009a; Smith et al., 2010), food intake (McGowan et al., 2005; McGowan et al., 2006), learning and memory (Ma et al., 2009a), and neuroendocrine function (McGowan et al., 2008; McGowan et al., 2009). The aim of this article is to provide a concise review of the central relaxin-3 system after the first decade of research into its biology. Despite early challenges, such as difficult synthetic chemistry of relaxin-3 and analogues (Bathgate et al., 2006b), recent progress has been assisted by the development of selective chimeric agonist and antagonist peptides (Liu et al., 2005a; Kuei et al., 2007). Similarly, the development of suitable transgenic mouse lines (relaxin-3 knockout mice) (Smith et al., 2009b; Sutton et al., 2009) and additional viral-based tools (Callander et al., 2009) provide a capacity to probe the roles of relaxin-3 in arousal, stress responses, cognition, metabolic homeostasis, circadian activity and sleep.

2. Discovery and characteristics of the neuropeptide, relaxin-3

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et al., 2003) and cardiac protective (Teerlink et al., 2009) agent. In contrast, ‘relaxin-3’ is a neuropeptide with a distinct profile of central physiological functions, and offers its own considerable therapeutic potential. Relaxin-3 is a 5 kDa peptide that consists of two chains (termed ‘A’ and ‘B’) and three disulphide bonds (two interchain and one intrachain), which is characteristic of its relaxin/insulin superfamily of peptides (Bathgate et al., 2002a; Liu et al., 2003b) (Fig. 1A). The relaxin-3 gene encodes a prohormone sequence, which like several other members of the superfamily contains a C-peptide that is removed by proteolytic processing (Bathgate et al., 2002a; Sherwood, 2004). All members of the ‘relaxin’ peptide family, including relaxin-3, contain the characteristic sequence ‘RXXXRXX(I/V)’ within the B-chain, which is essential for binding to the different cognate relaxin receptors (Bullesbach and Schwabe, 2000; Bathgate et al., 2002a, 2006a). Relaxin-3 is recognised as the ‘ancestral’ member of the relaxin peptide family, which emerged prior to the divergence of fish. The relaxin-3 gene is also highly conserved across species including fish, frogs, rodents and primates, suggesting the peptide product performs important physiological functions, as strong evolutionary pressure has prevented tolerability of changes/mutations in its structure (Bathgate et al., 2002b; Wilkinson et al., 2005a; Callander and Bathgate, 2010).

3. Distribution of relaxin-3 neurons in the brain Although limited peripheral expression of relaxin-3 has been reported (Liu et al., 2003b), the main site of relaxin-3 mRNA expression is the brain, where it has been detected in high concentrations in species including the zebrafish (Donizetti et al., 2008), mouse (Bathgate et al., 2002a; Smith et al., 2010), rat (Burazin et al., 2002; Tanaka et al., 2005), macaque (Ma et al., 2009b), and human (Liu et al., 2003b). Expression in brain is neuronal in nature and ultrastructural examinations of rat brain

Relaxin-3 was discovered in 2001 by searching for homologues of the relaxin hormone gene in the Celera Discovery System and Celera Genomics associated databases (Bathgate et al., 2002a). ‘Relaxin’ (designated relaxin-2, or H2 relaxin in humans) is largely characterised as a peripheral hormone that plays vital physiological roles in pregnant mammals, such as promoting growth and softening of the cervix and the development of the mammary apparatus (Sherwood, 2004). Relaxin has also demonstrated significant therapeutic potential as an anti-fibrotic (Samuel

Fig. 1. Schematic diagrams of (A) relaxin-3 and (B) RXFP3 (kindly provided by Dr Gabrielle Callander and Dr Daniel Scott, Florey Neuroscience Institutes).

Fig. 2. Relaxin-3 expressing neurons in the nucleus incertus of mouse brain. A high magnification image of a representative, nuclear fast red counterstained coronal brain section, illustrating X-GAL staining of LacZ reporter-gene expression in the ipsilateral nucleus incertus of a relaxin-3-knockout/LacZ-knockin mouse. Relaxin3-associated LacZ reporter-gene expression reflected by the presence of blue precipitate is visible in a cluster of NI neurons within their soma and proximal extensions.

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Table 1 Distribution of relaxin-3-like immunoreactivity, and RXFP3 mRNA and binding sites in adult rat brain. Brain region

Area/nucleus

RLN3-LI1,2

RXFP3 mRNA1,3

([125I] R3/I5) binding1,3

Forebrain

Claustrum Frontal cortex Insular cortex Medial/Orbital prefrontal cortex Parietal cortex Perirhinal cortex Retrosplenial cortex Secondary motor cortex Temporal cortex Visual cortex

+++/+ + nd + nd nd ++ + nd nd

+/ ++ ++ +(+) +++ ++ +/ + + +

+     +/  ++ ++ ++

Olfactory

Anterior olfactory n. Dorsal endopiriform n. Ependymal (sub-) layers, olfactory ventricle N. lateral olfactory tract Olfactory bulb Piriform (olfactory) cortex

+ ++ ++ ++ + +/

+++/+ +/ ++ + +++ ++

+++ +/ +/ +/ +++ 

Hippocampus

Alveus CA1/CA2 CA3 Dentate gyrus Entorhinal cortex Pre/Para/Post-subiculum

++(+) +++ ++/++++ ++++ ++ +(+)

+(+) + ++ +++ ++ +

  + + +/ 

Amygdala

Amygdalohippocampal area Basolateral n. Basomedial n. Central n. Cortical/Medial/Lateral n. Bed n. of stria terminalis

+++ +/ + + + ++

+++  +++ +++ +(+) ++++

++ +/ +/ ++ +(+) ++

Septum

Lateral n. Medial n. N. of diagonal band Septofimbrial n. Septohypothalamic n. Triangular n.

++++ ++++ +++ +++ + +/

+++ +++ ++ ++ ++++ +++

+     +/

Basal ganglia

Nucleus accumbens (shell) Substantia innominata Substantia nigra pars compacta Ventral tegmental area

+ ++ ++ (cb) ++/+

+ +/ + nd

   

Thalamus

Anterodorsal n. Anteromedial n. Anteroventral n. Central medial/lateral n. Habenular n. Intergeniculate leaflet Lateral/Medial geniculate n. Mediodorsal n. (lateral/paralaminar) Parafascicular n. Paratenial n. Paraventricular n. Peripeduncular n. Posterior intralaminar n. Reuniens n. Rhomboid n. Zona incerta

++ +++ ++ +(+) ++ ++/++++ ++ + nd +/ ++ ++ ++ ++ + +

 +/  +++ ++ ++ + +/ + +/ +++ ++ ++ ++ + +/

+/ +/ +/ ++ ++    +/  +++     

Hypothalamus

Anterior hypothalamic n. Arcuate n. Dorsomedial n. Lateral hypothalamic area Lateral mammillary n. Lateral preoptic area Lateroanterior n. Magnocellular preoptic n. Medial mammillary n. Medial preoptic area Medial tuberal n. Paraventricular n. Periventricular n. Posterior hypothalamic n. Premammillary n. Suprachiasmatic n.

+ +/+++ ++ ++++ + ++ ++ ++ +/+++ + ++ + + ++ ++ 

++ ++ ++ +++ nd +++ + +/ nd +++ ++ +++ +++ + nd +/

 +/  +        +++ +   

C.M. Smith et al. / Journal of Chemical Neuroanatomy 42 (2011) 262–275

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Table 1 (Continued ) Brain region

Brainstem

Area/nucleus

RLN3-LI1,2

RXFP3 mRNA1,3

([125I] R3/I5) binding1,3

Supramammillary n. Supraoptic area Ventromedial n.

+++ + +

nd +++ ++

 ++ 

Anterior pretectal n. Caudal linear n. Cuneiform n. Dorsal raphe´ n. Dorsal tegmental n. (capsule) Edinger-Westphal n. Gigantocellular reticular n. Inferior colliculus Inferior olive Interfascicular n. Interpeduncular nucleus Laterodorsal tegmental area Median raphe´ n. N. incertus N. prepositus N. raphe´ magnus N. raphe´ pontis N. solitary tract Paramedian raphe´ n. Periaqueductal grey Pontine central grey Pontine n. Pontine reticular n. Raphe´ interpositus n. Rostral linear n. Superior colliculus Supragenual n. Trigeminal n. spinal

+ + nd +++ +/ + nd + + ++ +++ nd + ++++ (cb) + nd ++ (cb) +/ nd ++ (cb) ++ (cb) + + + + + + nd

+ ++ + ++ +/ +/ +/ ++ +/ + ++++ + +/ ++++ +  nd ++ +/ ++ +/  +  + +++ +/ ++

  +/ + nd  nd ++ nd  +++   ++ nd nd nd ++  ++ nd   nd  +++ nd ++

Data is a summary from three studies: 1. Ma et al. (2007); 2. Tanaka et al. (2005); 3. Sutton et al. (2004). Scale: ++++, very dense; +++, dense; ++, moderate; +, sparse; , not present; nd, not determined. Regions with minimal expression were excluded for brevity. If the expression pattern or the density described was different in the cited papers, the higher of the two values is provided; if the expression pattern is more varied, both values are represented. Abbreviations: cb, cell bodies; n., nucleus (nuclei); RLN3-LI, relaxin-3-like immunoreactivity.

have revealed relaxin-3-like immunoreactivity (IR) present within the rough endoplasmic reticulum and Golgi apparatus at the cell soma, and within dense-core vesicles adjacent to the synapse in nerve terminals (Tanaka et al., 2005; Ma et al., 2009a). These features suggest that relaxin-3 is released into the synaptic cleft to act as a neurotransmitter. The neuroanatomical distribution of relaxin-3 positive cells has been comprehensively studied in adult rat (see Table 1) and mouse brain (Fig. 2), revealing intense expression in a tight cluster of neurons in a region known as the ‘nucleus incertus’, which lies in the midline periventricular central grey at the coronal level of the tegmentum (pons) (Bathgate et al., 2002a; Burazin et al., 2002; Tanaka et al., 2005; Ma et al., 2007; Smith et al., 2010; see Ryan et al., 2011 for review). Smaller, more diffuse populations of relaxin-3 neurons are present in the pontine raphe´ nucleus, the anterior periaqueductal grey (medial, ventral and lateral), and in an area dorsal to the substantia nigra (Tanaka et al., 2005; Ma et al., 2007; Smith et al., 2010). In the adult rat brain, the nucleus incertus was estimated to contain 2000 relaxin-3-positive neurons, while there were estimated to be 350 cells in the pontine raphe´ nucleus, 550 cells in the anterior periaqueductal grey, and 350 cells in the area dorsal to the substantia nigra (Tanaka et al., 2005). In the rat, the neuroanatomical distribution of pontine relaxin-3 positive neurons closely resembles the topography of the GABAergic nucleus incertus (Goto et al., 2001; Olucha-Bordonau et al., 2003), suggesting that relaxin-3 represents a good neurochemical ‘marker’ for the nucleus incertus, with relaxin-3 neurons tightly packed within the midline pars compacta (NIc) and more diffuse within the more lateral pars dissipata (NId) (Tanaka et al., 2005; Ma et al., 2007). Within the mouse brain, however, the distribution pattern of relaxin-3 positive neurons is not as well aligned with that of the nucleus incertus (also designated ‘nucleus

O’) across its rostral-caudal extent, and a larger percentage of relaxin-3 negative neurons within this region suggests relaxin-3 may not be as reliable as a marker for nucleus incertus cells in this species (Smith et al., 2010). Interestingly, in both species a small number of relaxin-3 neurons were also detected just caudal to the nucleus incertus immediately adjacent to the 4th ventricle, but these neurons have not been designated as constituting a separate population. In other anatomical studies, relaxin-3 mRNA expression was observed in zebrafish in a cluster of cells in the midbrain periaqueductal grey, and in a more posterior cell group, which is a likely equivalent of the rodent nucleus incertus (Donizetti et al., 2008; Donizetti et al., 2009). Although a primate equivalent of the rodent nucleus incertus is yet to be formally characterised, recent studies in the macaque (Macaca fascicularis) detected relaxin-3 mRNA and relaxin-3 IR in large, elongated neurons within the ventromedial central grey at the level of the tegmentum, while sparse relaxin-3 positive cells were also detected in the periaqueductal grey (Ma et al., 2009b), representing populations of relaxin-3 neurons homologous to those detected in the equivalent regions in the rodent (Ma et al., 2007; Smith et al., 2010). In another recent study in the Rhesus macaque (Macaca mulatta), relaxin-3-like IR was reportedly detected within cell bodies in the cerebellar cortex, regions of the dorsal and lateral tegmental nucleus, and in the ventral cochlear nucleus (Silvertown et al., 2010). In this same study, relaxin-3-like IR was reported in cell bodies in the dorsal raphe´ nucleus, pontine reticular nucleus, and regions of the dorsal and ventral tegmental nuclei (Silvertown et al., 2010), although the authenticity of this immunostaining has not be independently validated and the presence of relaxin-3 mRNA was not described in these areas. In fact, despite the use of

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an earlier characterised antiserum (see Tanaka et al., 2005), these authors did not describe the abundance of relaxin-3 immunoreactive nerve fibres observed in rat (Tanaka et al., 2005), mouse (Smith et al., 2010) and non-human primate (Ma et al., 2009b), possibly due to differences in tissue fixation and processing. Nonetheless, it will be important to eventually describe the precise distribution of all relaxin-3 producing neurons and their projections in the human brain. 4. Neurochemistry of relaxin-3 neurons Following the localisation of relaxin-3 in the nucleus incertus, double-label immunohistochemical studies confirmed that relaxin-3 neurons were GABAergic, reflected by their co-expression of the GABA synthesising enzyme, GAD (Ford et al., 1995; OluchaBordonau et al., 2003; Ma et al., 2007). Detailed anatomical studies indicate that these GABAergic neurons have prominent long projections throughout the brain, rather than representing a population of locally projecting GABA interneurons (Ford et al., 1995; Goto et al., 2001; Olucha-Bordonau et al., 2003). Several other peptides and proteins have been observed within the nucleus incertus, including neuromedin B (or ‘ranatensin-like peptide’) (Chronwall et al., 1985; Wada et al., 1990), cholecystokinin (Kubota et al., 1983), calbindin and calretinin (Paxinos et al., 1999) and the metabolising enzyme, acetylcholinesterase (OluchaBordonau et al., 2003). Although double labelling experiments are yet to confirm the presence of these transmitters and other markers in relaxin-3 neurons, a degree of co-expression seems likely due to the high density/proportion of relaxin-3 neurons within the nucleus (see, e.g. Ma and Gundlach, 2007; Ryan et al., 2011). Further studies are also required to examine the neurochemical fingerprint of the relaxin-3 neurons present in areas other than the nucleus incertus. Careful analysis of the receptors expressed by relaxin-3 neurons is another important goal, as it will provide insights into the neural inputs these cells receive and the central pathways and stimuli that might regulate relaxin-3 production and release. In this regard, nucleus incertus relaxin-3 neurons in the rat express type 1 corticotropin-releasing factor receptors (CRF1) (Bittencourt and Sawchenko, 2000; Tanaka et al., 2005), and respond with increased c-Fos and relaxin-3 expression following icv CRF injections or different physiological stressors (Tanaka et al., 2005; Banerjee et al., 2010). Relaxin-3 neurons in the rat nucleus incertus express 5HT1A serotonin receptors (Miyamoto et al., 2008), and receptors for orexins/hypocretins (OR1 and OR2) are present within a region corresponding to the NI (Greco and Shiromani, 2001). The implications of the receptor profile of relaxin-3 positive neurons for the functional role of this system are further discussed below (see Section 9). 5. The relaxin-3 receptor, RXFP3 The cognate receptor for relaxin-3 is relaxin family peptide 3 receptor (RXFP3) (Fig. 1B) (Bathgate et al., 2006a). Also known as G-protein coupled receptor 135 (GPCR135) (Liu et al., 2003b), it was initially discovered in 2000 by screening a human cerebral cortex cDNA library for G-protein coupled receptors and named as SALPR, the somatostatin- and angiotensin-like peptide receptor, because it shared similarity with the somatostatin receptor SSTR5 and the angiotensin AT1 receptor (Matsumoto et al., 2000). While relaxin-3 was initially thought to be another endogenous ligand for the recently discovered relaxin receptors (Hsu et al., 2002), soon after its discovery, relaxin-3 was identified as a high affinity ligand for GPCR135 (Liu et al., 2003b). Although in cell-based studies relaxin-3 is capable of binding and activating three related Gprotein coupled receptors – RXFP3, RXFP1 (previously known as

LGR7) (Sudo et al., 2003), and RXFP4 (previously GPCR142) (Liu et al., 2003a) – considerable evidence suggests that RXFP3 is the native receptor for relaxin-3. For example, RXFP3 displays high affinity for relaxin-3, and the genes encoding both proteins appear to have phylogenetically co-evolved with each other (Liu et al., 2003b; Wilkinson et al., 2005b). Furthermore, while a strong overlap exists between the distribution of relaxin-3-positive fibres and RXFP3 mRNA/binding sites, RXFP4 is mainly expressed within the gut and is largely absent from the brain (Sutton et al., 2005) (in fact, RXFP4 is a pseudogene in rat; Chen et al., 2005); and although RXFP1 is expressed within the brain, it is present within regions largely devoid of relaxin-3 fibres. Finally, relaxin-3 is the only member of the relaxin/insulin superfamily that can activate RXFP3 (Liu et al., 2003b), whereas relaxin is the preferred ligand for RXFP1 (Sudo et al., 2003) and insulin-like peptide 5 (INSL5) is the endogenous ligand for RXFP4 (Liu et al., 2005b; Sutton et al., 2005; see Bathgate et al., 2006a; Callander and Bathgate, 2010 for review). Studies in vitro using non-neuronal cells that over-express RXFP3 have shown that it couples to inhibitory G-proteins and inhibits cAMP accumulation (Liu et al., 2003b). Later studies revealed that relaxin-3 bound RXFP3 also activates the extracellular signal-regulated kinase (ERK) 1/2 pathway, and that ERK1/2 phosphorylation is downstream of the activation/recruitment of Gi/o proteins, which seems to require RXFP3 internalisation and/or compartmentalisation into lipid-rich environments. ERK1/2 activation predominantly occurs via activation of a protein kinase Cdependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase are also involved to a lesser extent (van der Westhuizen et al., 2007). These signalling aspects are difficult to study in the intact nervous system due to the low density of endogenous receptors, but alternate amplification methods including calcium imaging or reporter assays that have recently been used to describe other peptide and receptor systems in vivo or in intact brain slices (e.g. Garaschuk et al., 2006; Justice et al., 2008; Yamanaka and Tsunematsu, 2010) may lead to comparable insights in the future. 6. Distribution of RXFP3 expressing neurons within the brain The distribution of RXFP3 mRNA and binding sites has been investigated in the rodent brain using in situ hybridisation and radioligand binding respectively, revealing a regional pattern of distribution which largely overlaps that observed for relaxin-3 positive fibres (Sutton et al., 2004; Sutton et al., 2005; Ma et al., 2007; Smith et al., 2010) (Table 1; Fig. 3). Areas of particular functional interest that are rich in relaxin-3 fibres and RXFP3 mRNA/binding sites include preoptic, periventricular and lateral hypothalamus, and the paraventricular and supraoptic nuclei; the septum, hippocampus, and other major nodes of the septohippocampal pathway such as the median raphe´, interpeduncular and supramammillary nuclei; the superior colliculus and the periaqueductal grey; and the extended amygdala, including the bed nucleus of the stria terminalis and the central and medial amygdala. Interestingly however, in some brain regions the distributions of relaxin-3 fibres and RXFP3 mRNA/binding sites do not overlap. For example, a high density of relaxin-3-positive fibres has been observed within the midline cingulate and retrosplenial cortices in the rat and mouse brain, whereas RXFP3 mRNA is expressed by neurons in more lateral and ventral cortical regions and RXFP3 binding sites are not enriched in cingulate cortices (Sutton et al., 2004; Sutton et al., 2005; Ma et al., 2007; Smith et al., 2010). It is currently unknown whether these relatively rare ‘mismatches’ reflect differences in the detection thresholds involved in the methods employed for visualising relaxin-3 and RXFP3, or whether, for example, RXFP3 can be

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Fig. 3. Schematic illustration of the distribution and relative densities of relaxin-3-like immunoreactivity, RXFP3 mRNA and RXFP3 binding sites in coronal sections through the rostrocaudal extent of the C57BL/6J mouse brain. A series of coronal drawings, illustrate the distribution and relative density of relaxin-3 (fine red lines), RXFP3 mRNA (blue dots) and [125I]-R3/I5 binding sites (green areas). Scale bar, 1 mm. For more details see Smith et al. (2010). Reproduced with permission from Smith et al. (2010).

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Fig. 3. (Continued ).

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activated via volume transfer from distant release sites. Lastly, the distribution of RXFP3 in non-human primate or human brain has not been reported in detail (but see Ma et al., 2009c). The distribution of relaxin-3 and RXFP3 within specific brain regions/ circuits of rat and mouse brain is further described below, in relation to the putative functions of relaxin-3 signalling (see Section 9). Although the studies mentioned have revealed the regional and cellular distribution of RXFP3, attempts to determine the neurochemical ‘phenotype’ of RXFP3 expressing neurons has been hampered by the unavailability of suitable antibodies to directly and reliably detect RXFP3 protein in brain sections – a problem commonly encountered with peptide GPCRs. However, the recent production of mouse strains that express marker proteins such as eGFP and LacZ within RXFP3-positive neurons represents a viable alternative method for the detection of neuronal RXFP3 expression in the future. This will be important for understanding the function of relaxin-3 signalling and the mechanisms by which RXFP3 activation modulates physiology and behaviour. 7. Developmental expression of relaxin-3 and RXFP3 During development in the rat, low levels of relaxin-3 mRNA and peptide are detectable as early as E18 and at birth, respectively, in a region homologous to the adult nucleus incertus (Miyamoto et al., 2008). By P7, these levels of mRNA/peptide approach those observed in the adult, while relaxin-3 immunoreactive fibres are detectable within the hypothalamus at P1. Additional details of the distribution of relaxin-3 positive neurons throughout the development of the mouse brain are available in the open access Allen Brain Atlas (Lein et al., 2007), which reveals that relaxin-3 mRNA is detectable in a region analogous to the adult nucleus incertus as early as E15.5. The developmental profile of RXFP3 expression has been examined in mice (Boels et al., 2004) and Northern blot analysis detected RXFP3 transcripts from E11, while in situ hybridisation detected RXFP3 mRNA in the telencephalon and somites at E10.5. At later developmental stages, expression was present within distinct brain regions in a pattern similar to that observed in the adult (Smith et al., 2010). For example, by E16-E18 RXFP3 was detected within the hippocampus, thalamus, hypothalamus, pons, medulla and cerebral cortex (Boels et al., 2004). This pattern of embryonic expression corresponds with data available in the Allen Brain Atlas (Lein et al., 2007), where populations of cells expressing RXFP3 mRNA are clearly visible within the hypothalamus, thalamus, and hippocampus from E18.5, while expression within an area analogous to basal forebrain is evident as early as E13.5. Interestingly, both studies detail RXFP3 expression within the spinal cord at later developmental time points, however the distribution of RXFP3 in adult rodent spinal cord has not been investigated. 8. Pharmacological, genetic and viral tools for investigating relaxin-3 function The majority of studies investigating the in vivo function of relaxin-3/RXFP3 conducted to date have involved injections into rat brain of native relaxin-3 or a specific RXFP3 agonist or antagonist. Although not considered its cognate receptor, RXFP1 is likely to be activated by high concentrations of exogenous relaxin3 delivered icv (see, e.g. Bathgate et al., 2006b; Otsubo et al., 2010). Therefore, the development of a specific chimeric peptide RXFP3 agonist consisting of the relaxin-3 B-chain and insulin-like peptide 5 (INSL5) A-chain (termed ‘R3/I5’) (Liu et al., 2005a) and the subsequent development of a structurally related antagonist, R3(BD23–27)R/I5, has allowed more powerful functional studies

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(see Section 9). These peptides display high affinity/potency and binding selectivity at RXFP3 cf. other relaxin family peptide receptors (Liu et al., 2005a; Kuei et al., 2007) and most recently, more readily synthesised, simpler RXFP3 peptides have also been produced and tested in vivo (Haugaard-Kedstro¨m et al., 2011). The behavioural consequences of life-long relaxin-3 deficiency have been investigated using relaxin-3 knockout (KO) mice (Smith et al., 2009b; Sutton et al., 2009). Although interesting insights have been made, some initial reports on the behavioural phenotype of mixed background (129S5:B6) relaxin-3 KO mice were conflicting, likely due to one or more of the inherent disadvantages in using such mixed background mice. Recently, however, more reliable data has been obtained using relaxin-3 KO mice after >10 generations of backcrossing onto a C57BL/6J genetic background (see, e.g. Smith et al., 2009a; Dugovic et al., 2010). An alternative experimental approach to study the effects of relaxin-3 deficiency (that avoids the possibility of developmental compensation that can occur with the conventional whole-of-life gene knockout approach), is the use of virally-delivered micro-RNA to deplete or ‘knock-down’ relaxin-3 mRNA and peptide from relaxin-3 neurons. In this regard, our laboratory has recently developed an efficient AAV1/2-vector that drives long lasting neuronal expression of specific micro-RNA targeting the 30 untranslated region of relaxin-3 mRNA, which when injected into the nucleus incertus of the rat eliminates >80% relaxin-3 mRNA from the region and almost completely depletes relaxin-3-like IR in forebrain regions such as the septum for 3 weeks (Callander et al., 2009). 9. Putative functional roles of relaxin-3/RXFP3 signalling based on anatomical and experimental evidence Following the discovery that relaxin-3 was highly expressed within the nucleus incertus, speculation about the possible functional role of relaxin-3 within the brain was initially based on two anatomical studies describing the efferent and afferent connections of the rat nucleus incertus (Goto et al., 2001; OluchaBordonau et al., 2003). Both studies suggested the nucleus incertus formed part of a ‘behavioural activation’ (or arousal) network, based on strong associations between nucleus incertus and other regions with established roles in such processes, such as the median raphe´ nucleus. This association was reflected by high levels of reciprocal neuronal connections between the nucleus incertus and median raphe´, and largely common, ascending projection patterns to regions/circuits involved in functions such as circadian rhythm and sleep/wake states, memory and spatial navigation, and the response to stress (see Ryan et al., 2011 for review). More recently, the distribution of relaxin-3 fibres and RXFP3 has been comprehensively mapped within the rat and mouse brain (and to a lesser extent in other species), and a number of functional studies have demonstrated the ability of exogenous relaxin-3 to modulate behaviours that are conferred by circuits which are rich in relaxin-3/RXFP3. Taken together, this knowledge is consistent with the theory that the relaxin-3/nucleus incertus system represents an ascending arousal system. This theory is based on the anatomical similarities between the relaxin-3/nucleus incertus system and other well characterised ascending arousal systems, such as the monoamine 5-HT/raphe´, histamine/tuberomammillary and noradrenaline/locus coeruleus pathways, and the peptidergic orexin network seated in the lateral hypothalamus (see, e.g. Jones, 2005; Saper et al., 2010). All of these systems modulate arousal – which encompasses control over sleep/wake states, and a range of ‘hyper-wakeful’ functions such as attention, motivation, working memory and navigation that are required for an animal to be awake and alert. Although not their primary function, arousal systems are also able to modulate feeding/metabolism and stress

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responses. As a growing number of experimental studies support the theory that relaxin-3 can act as an arousal transmitter, it is our view that subsequent functional reports should describe their findings on relaxin-3 in terms of a more integrated theory of relaxin-3 function (see also Kastin and Pan, 2010). A broad criticism of existing functional studies is that while they have been informative, findings are usually discussed in terms of isolated relaxin-3 circuits, rather than interpretation in terms of overall relaxin-3 function. For example, several groups have observed that central infusion of high doses of exogenous relaxin-3 is orexigenic in rats, and have postulated a major primary role of endogenous relaxin-3 signalling is in the control of feeding and metabolism, citing the presence of relaxin-3 fibres within hypothalamic ‘feeding centres’. However, when examined from an integrated standpoint, levels of relaxin-3 within major feeding centres are relatively low, compared with the broader innervation density throughout the brain. A similar ‘restricted’ view has been observed previously in the field with, for example, the neuropeptide orexin, initially thought to primarily modulate feeding/ metabolism based on the orexigenic response elicited by its central infusion, whereas it is now considered primarily as an arousal transmitter (Sutcliffe and de Lecea, 2002; Sakurai, 2007; Yamanaka and Tsunematsu, 2010). Thus, some putative modulatory roles of relaxin-3 are discussed, and neuroanatomical and functional evidence is presented within an integrated framework of a broadly influential ascending arousal system. 9.1. Relaxin-3 neurons: an ascending arousal system The concept of ascending arousal systems – structures within the brain stem promoting forebrain wakefulness – was first postulated in 1916 following observations that viral-induced lesions at the junction of the midbrain and diencephalon resulted in excessive sleep and lethargy (Von Economo, 1930). Since then, several individual (but highly interconnected) ascending arousal systems have been defined, including populations of neurons which express 5-HT, noradrenaline, acetylcholine, dopamine, histamine and orexin within the dorsal and median raphe´, locus coeruleus, basal forebrain and pedunculopontine/laterodorsal tegmental nuclei, periaqueductal grey, tuberomammillary nucleus, and lateral hypothalamus, respectively (Jones, 2005; Saper et al., 2010). These pathways promote wakefulness (including increased cortical activation) via direct connections with the cortex and indirect connections with thalamic relay neurons; and also via direct inhibitory connections with the ventrolateral preoptic nucleus (often referred to as the ‘sleep switch’). These arousal pathways are also involved in modulating a range of processes and behaviours associated with an alert and active animal, including hippocampal-dependent attention and spatial memory, due to projections onto circuits such as the septohippocampal system that underlie these functions (e.g. Gerashchenko et al., 2001; Smith and Pang, 2005). Similarly, arousal pathways can modulate motivation/reward, the response to stress and feeding behaviour via interactions with the mesolimbic dopaminergic pathway, CRFassociated nuclei and hypothalamic feeding centres, respectively (e.g. Winsky-Sommerer et al., 2004; Harris et al., 2005). The neuroanatomical distribution of the relaxin-3/nucleus incertus system displays a strong similarity to the existing, characterised ascending arousal systems. The nucleus incertus lies just dorsal and caudal to the median raphe´ and medial to locus coeruleus, and the pattern of relaxin-3 positive efferent projections largely overlaps that of these nuclei and other ascending arousal pathways, for example featuring a strong innervation of the septohippocampal pathway. Therefore, the relaxin-3/nucleus incertus system likely acts in parallel with other arousal systems. Furthermore, relaxin-3 positive fibres and RXFP3 are present

within brain regions in which populations of arousal-related neurons are located (apart from the locus coeruleus), suggesting that relaxin-3 also likely acts in series with the majority of other arousal systems. Additionally, several studies have provided evidence that relaxin-3 neurons receive inputs from other arousal pathways. Relaxin-3 positive cells within the nucleus incertus express the 5-HT1A receptor, and 5HT depletion by p-chlorophenylalanine (300 mg/kg, i.p., 72, 48 and 24 h before), produced a significant increase in relaxin-3 gene expression in the nucleus incertus (Miyamoto et al., 2008). Orexin receptors (OR1 and OR2) are also expressed within a region analogous to the nucleus incertus in the rat (Greco and Shiromani, 2001), and recent preliminary electrophysiological studies have revealed that orexin A and B can activate and increase the spontaneous action potential firing of neurons within the nucleus incertus (Blasiak et al., 2010). Various functional studies have begun to confirm the role of relaxin-3 in modulating processes and behaviours relevant to arousal, which are predicted by the neuroanatomical distribution of relaxin-3 and RXFP3. These behaviours can be broadly classified as: (i) hippocampal theta rhythm-dependent behaviours such as spatial memory; (ii) response to stress; (iii) feeding and metabolism; (iv) control of sleep/wake states and circadian rhythm and; (v) motivation and reward. 9.2. Hippocampal theta rhythm-dependent behaviours Hippocampal theta rhythm is a synchronous oscillation of brain electrical activity (between 4 and 8 Hz in humans, 5 and 10 Hz in rodents), which has long been associated with hippocampal output and dependent functions such as REM sleep and spatial memory (Oddie and Bland, 1998; Bland and Oddie, 2001; Buzsaki, 2005; Vertes, 2005). Numerous regions within the brain contribute to the control of hippocampal theta rhythm and are collectively referred to as the septohippocampal pathway (or system) (Vertes and Kocsis, 1997). Importantly, high levels of relaxin-3/RXFP3 are present within the septum (known as the theta ‘pacemaker’), the hippocampus itself, and within all the major nodes of the septohippocampal pathway such as the median raphe´, interpeduncular nucleus, posterior hypothalamus and supramammillary nucleus (see Table 1). Studies in rats have established that the nucleus incertus can modulate hippocampal theta rhythm. Electrical stimulation of the nucleus incertus in anaesthetised rats induces theta rhythm, while electrolytic lesions and GABAergic inhibition block the ability of the brainstem region, reticularis pontis oralis, to generate theta (Nunez et al., 2006). Recent studies have also demonstrated the ability of relaxin-3 signalling to modulate theta rhythm. Injections of the RXFP3 agonist R3/I5 into the medial septum of anaesthetised rats produced an increase in the power of the hippocampal theta frequency band, while in conscious rats a similar infusion promoted theta rhythm in a home cage environment; and septal injections of an RXFP3 antagonist reduced hippocampal theta rhythm in an environmentally enriched behavioural arena (Ma et al., 2009a). Wakeful hippocampal theta rhythm is required for many behavioural processes including spatial memory, and septal injection of an RXFP3 antagonist impaired the performance of rats in a spatial memory test, the spontaneous alternation task (conducted in a plus maze) (Ma et al., 2009a). These findings indicate that endogenous relaxin-3 signalling within the medial septum promotes spatial memory and exploratory activity in the rat. Such a role is further supported by the presence of a substantial relaxin-3 innervation of regions involved in spatial orientation and navigation such as the superior colliculus and cingulate/retrosplenial cortex (see Table 1). In sleeping mammals, hippocampal theta rhythm is associated with REM sleep, and a recent preliminary report indicated that

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backcrossed C57BL/6J relaxin-3 KO mice exhibited more frequent REM sleep episodes and increased REM sleep duration over the entire light-dark cycle than wild type (WT) mice (Dugovic et al., 2010). In line with these findings, icv infusion of an RXFP3 antagonist in rats reportedly produced a prolongation of REM sleep time over 6 h post-administration (Dugovic et al., 2010). These data suggest endogenous relaxin-3 may act ‘globally’ to reduce the occurrence of REM sleep and associated theta across the circadian cycle, while the local infusion studies indicate that elevated relaxin-3 signalling in medial septum promotes theta. These apparently contradictory findings may be due to differences in ‘global’ versus ‘local’ manipulation of RXFP3 activation, but may also indicate that relaxin-3 signalling can promote or inhibit hippocampal theta rhythm, depending on the current or prior behavioural state (e.g. awake or sleeping) of the animal. Clearly this interesting issue requires further investigation. 9.3. Response to stress Relaxin-3 neurons within the nucleus incertus express high densities of CRF1 receptors, and relaxin-3 fibres and/or RXFP3 mRNA/binding sites are highly concentrated within brain ‘stress centres’ including the extended amygdala (amygdala/bed nucleus stria terminalis) and paraventricular hypothalamic nucleus (Bale and Vale, 2004; Neufeld-Cohen et al., 2010). In line with a role in the stress response, icv CRF and psychogenic stressors have been shown to activate relaxin-3 expressing cells, while stressors (e.g. swim stress or water immersion-restraint) have been shown to increase relaxin-3 mRNA expression in the nucleus incertus – an effect blunted by icv injection of a CRF1 antagonist (Tanaka et al., 2005; Banerjee et al., 2010). Furthermore, following icv relaxin-3 administration c-Fos and CRF expression were increased in the paraventricular nucleus of the hypothalamus 1–2 h after administration (Watanabe et al., 2010), while significant increases in plasma ACTH, corticosterone and prolactin levels were also detected (McGowan et al., 2007a; Watanabe et al., 2010). Additionally, injection of a specific RXFP3 agonist into the central amygdala reduced the characteristic freezing response rats display following fear conditioning (Ma et al., 2010). Taken together, these data strongly imply an involvement of relaxin-3 in stress responses, at least partly through interactions with CRF. Interestingly however, chronic injections of relaxin-3 into the paraventricular nucleus (180 pmol 2 daily, 7 days) did not produce a significant change in plasma prolactin or corticosterone when measured on day 8 (McGowan et al., 2006), indicating possible differential acute vs chronic effects of the peptide. Further studies are required to assess these possibilities. (Note: A recent minireview on relaxin-3 addresses its putative functions in the hypothalamus; Tanaka, 2010.) 9.4. Feeding and metabolism Reports of the presence of high densities of RXFP3 within various hypothalamic ‘feeding centres’, such as the hypothalamic paraventricular nucleus (Liu et al., 2003b), prompted a series of experiments which measured the orexigenic response of rats to central relaxin-3 infusions. Human relaxin-3 (54 and 180 pmol) injected icv into rats significantly increased food intake in satiated rats within the first hour after administration, in both the early light and dark phases (McGowan et al., 2005). Furthermore, icv infusion of the RXFP3 agonist R3/I5 both acutely and chronically increased feeding, and this effect was blocked by an RXFP3 antagonist, R3(BD23–27)R/I5 (Kuei et al., 2007); while centrally administered human relaxin, which interacts with RXFP1 but not RXFP3, decreased food intake in the early dark phase (McGowan et al., 2010) – strengthening the case for RXFP3 mediated effects on feeding.

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Regions within the hypothalamus that confer these orexigenic effects were also investigated, and human relaxin-3 promoted food intake when locally injected into the paraventricular, supraoptic and arcuate nuclei and into the anterior preoptic area (McGowan et al., 2007b). However, the nature of any interactions with other hypothalamic peptides remain unclear, as no significant differences were observed in the hypothalamic expression of neuropeptide Y, proopiomelanocortin or agouti-related peptide mRNAs following acute icv relaxin-3 (McGowan et al., 2005; McGowan et al., 2009). Therefore, further time course studies of any changes in hypothalamic feeding-related peptide mRNA or peptide levels after relaxin-3 administration are required to understand the precise sites of action. In this respect, chronic twice daily administration of human relaxin-3 (180 pmol) for 7 days produced a significant increase in cumulative food intake and plasma leptin, and a trend for an increase in body weight and epididymal fat mass in rats, but no significant differences were detected in intrascapular brown adipose tissue, or measures of energy expenditure such as VO2, RER (respiratory exchange ratio) and physical activity (McGowan et al., 2006). In a separate study, human relaxin-3 was administered at 600 pmol/day for 14 days via osmotic minipumps, which produced a significant increase in cumulative food intake and body weight, a significant increase in blood leptin and insulin levels and increased epididymal fat mass, but no difference in intrascapular brown adipose tissue (BAT) weight (Hida et al., 2006). In addition, chronic icv administration of the RXFP3 agonist R3/I5 for 2 weeks increased plasma insulin and leptin (Sutton et al., 2009). Despite the ability of high doses of centrally administered relaxin-3 to modulate feeding in rats, due to the broad neuroanatomical distribution of relaxin-3/RXFP3, it is likely that the modulation of feeding behaviour does not represent the primary endogenous role of relaxin-3. Rather, we suggest such orexigenic effects of exogenous relaxin-3 should be interpreted in terms of the putative broader function of relaxin-3 as an arousal neuromodulator. Finally, with respect to water drinking behaviour, a recent study demonstrated significant increases in water intake following icv injection of 54–540 pmol relaxin-3 in rats (Otsubo et al., 2010). Predictably however, this effect was similar to that seen after icv relaxin (Summerlee and Robertson, 1995; Sunn et al., 2002), and was likely due to activation by human relaxin-3 of RXFP1 in the subfornical organ (Sunn et al., 2002; Ma et al., 2006). These data emphasize the importance of employing selective RXFP3 agonists in functional experiments. 9.5. Control of sleep/wake states and circadian rhythm The distribution of relaxin-3/RXFP3 strongly suggests this system is able to modulate sleep/wake states and circadian rhythm. In particular, projections to the lateral hypothalamus predict possible interactions with the orexin and melanin concentrating hormone (MCH) systems, which play a role in stabilising sleep/wake states. Furthermore, relaxin-3/RXFP3 elements are present within the intergeniculate leaflet and suprachiasmatic nucleus (often referred to as the ‘circadian pacemaker’), which receive photic inputs from the retina and are involved in integrating photic and non-photic sensory information (Harrington, 1997; Morin and Allen, 2006). In line with this, preliminary studies have observed that relaxin-3 mRNA expression varies over the 24 h circadian cycle, with a peak at 2000 h (during the dark/ active phase) and a trough at 0800 h (during the early light/ inactive phase) (Banerjee et al., 2006), and that RXFP3 activation alters the activity of IGL neurons in vitro (Blasiak et al., 2009). Whether relaxin-3 contributes to the generation of circadian rhythm, or is ‘down-stream’ of these essential network processes, is currently unknown.

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Despite compelling anatomical evidence suggesting a role of relaxin-3 in the control of sleep/wake states and circadian rhythm, to date these roles have only been tested in a limited number of preliminary studies. For example, icv infusion of the RXFP3 agonist R3/I5 increased wakefulness (Dugovic et al., 2010), and similar infusions have been shown to increase wakeful behaviours such as locomotor activity/exploratory behaviour (Sutton et al., 2009), possibly indicative of increased general arousal. These findings are consistent with recent studies conducted using two separate C57BL/6J backcrossed cohorts of relaxin-3 KO mice, whereby relaxin-3 deficiency led to increased time spent sleeping during the dark (‘active’) phase (Smith et al., 2009a; Dugovic et al., 2010). However, a putative primary role of relaxin-3 in arousal predicts that future functional studies will confirm an influence of relaxin-3 on the control of sleep/wake states and circadian rhythm. 9.6. Motivation and reward The final common ‘reward pathway’ within mammalian brain consists of dopamine neurons in the ventral tegmental area that project to the nucleus accumbens. This mesolimbic dopaminergic pathway is activated when an animal engages in pleasurable behaviour such as eating food or mating, and is also involved in the motivational drive to engage in such behaviour (e.g. Nestler and Carlezon, 2006; Ikemoto, 2010). Neuroanatomical data suggests that relaxin-3/RXFP3 signalling may impinge directly or indirectly on the mesolimbic dopaminergic pathway and related limbic regions including the amygdala. In line with this, we observed that backcrossed C57BL/6J relaxin-3 KO mice run less distance on voluntary running wheels compared to WT littermate controls (suggesting reduced motivation) (Smith et al., 2009a); while in a separate preliminary study relaxin-3 KO mice were observed to eat less palatable food (suggesting anhedonia, or a diminished ability to experience pleasure from normally rewarding activities) (Shoblock et al., 2010). 9.7. Other modulatory roles in reproduction and metabolism? Some evidence suggests that relaxin-3 may play a modulatory role in reproduction or associated behaviour. For example, local infusion of relaxin-3 into the hypothalamic paraventricular nucleus increased plasma luteinising hormone levels, and this effect was blocked by pre-treatment with a peripheral GnRH antagonist (McGowan et al., 2006). Furthermore, relaxin-3 dosedependently stimulated the release of GnRH from the GT1–7 hypothalamic neuronal cell line, which expresses RXFP1 and RXFP3 (McGowan et al., 2008, 2009). Lastly, chronic R3/I5 treatment produced a significant elevation in plasma testosterone (Sutton et al., 2009). These possible interactions with GnRH neurons in the anterior hypothalamus/septum and subsequent influences upon the hypothalamic-pituitary-gonadal axis are of interest, considering the well defined role the relaxin hormone plays in reproduction (Sherwood, 2004). Therefore, further studies are warranted of the precise physiological role that relaxin-3/ RXFP3 signalling plays in reproductive behaviours. An interaction between relaxin-3 signalling and the hypothalamus-pituitary-thyroid axis is also possible, as local infusion of relaxin-3 into the hypothalamic paraventricular nucleus decreased plasma thyroid-stimulating hormone (TSH) levels (McGowan et al., 2006), and the large populations of TRH neurons in the hypothalamus may be direct or indirect targets of relaxin-3/ RXFP3 inputs. Interestingly, thyrotropin-releasing hormone degrading enzyme (TRH-DE) is highly expressed in the mouse nucleus incertus (Lein et al., 2007). TRH-DE is known to inactivate TRH (Wilk, 1986), and subsequently plays a role in controlling both TRH-induced TSH release and prolactin release in the anterior

pituitary (Cruz et al., 2008; Sanchez et al., 2009). It is possible, therefore, that the nucleus incertus is a target for TRH under certain physiological conditions. 10. Relevance of relaxin-3 to human disease Relaxin-3 deficiency or peptide or receptor gene polymorphisms have not been identified or linked to any clinical disease, but the broad range of behaviours that relaxin-3 putatively modulates suggests that drugs which target the relaxin-3 system offer potential as therapeutic agents for psychiatric or neuroendocrinerelated illnesses. For example, the ability of relaxin-3 to modulate theta rhythm highlights its relevance to a multitude of diseases including memory deficits, autism and schizophrenia; and an ability of drugs which target RXFP3 to modulate sleep architecture may be effective for the treatment of insomnia and a range of conditions in which normal sleep patterns are perturbed. An involvement in motivation, reward and feeding circuits suggests a therapeutic potential in drug addiction and feeding disorders, while a strong association with stress systems indicate relaxin-3/ RXFP3-based therapies may successfully treat anxiety disorders and/or depression. In this context, existing anatomical and functional evidence suggests that relaxin-3 is capable of modulating key processes/ behaviours which are often perturbed in clinical depression. Stress is a well known trigger for depression in humans, and is often associated with hyper-reactive stress responses and increased circulating stress hormones (Arborelius et al., 1999); and considerable evidence suggests relaxin-3/RXFP3 can modulate CRF/stress circuits and HPA axis activity. Depression is often associated with reduced mood and motivation (anhedonia); and relaxin-3/RXFP3 impinges on relevant limbic/mesocortical circuits (Nestler and Carlezon, 2006), while relaxin-3 KO mice display potential deficits in rewarding and motivated behaviours. Depression is often associated with body weight changes; and central infusion of relaxin-3 in rats is robustly orexigenic in rats. Insomnia or hypersomnia are common in depressed patients (Argyropoulos and Wilson, 2005); and relaxin-3/RXFP3 networks are distributed within circadian and arousal/sleep regions within the brain, while preliminary reports indicate that relaxin-3 KO mice and rats infused with an RXFP3 antagonist spend increased time sleeping. Depressed patients display robust elevations in REM sleep and other changes in brain activity related to hippocampal theta rhythm; and nucleus incertus associated relaxin-3/RXFP3 signalling can modulate hippocampal theta rhythm. Similar systems, such as the monoamine 5-HT and noradrenaline networks, form the target of almost all currently available pharmacological antidepressants, while similar neuropeptide systems, including orexin are attracting interest as exciting future targets. (Berton and Nestler, 2006; Ruhe et al., 2007; Werner and Covenas, 2010). 11. Future directions Several genetic and viral-based techniques are poised to facilitate relaxin-3 research in the future. While preliminary reports describing the characteristics of relaxin-3 KO mice have been informative, such ‘life-long’ gene/peptide deficits often result in compensation (obscuring phenotypes) and/or developmental abnormalities (resulting in phenotypes unrelated to ‘adult’ deficiencies in the target gene product). In the future, conditional relaxin-3 knockout mice could be produced, but the recent development of viral constructs that can ‘knock-down’ relaxin-3 mRNA in areas such as the nucleus incertus of adult rats by 80% for weeks/months (Callander et al., 2009) should help circumvent any such problems and better elucidate the functions of relaxin-3 neurons. Furthermore, our laboratory is currently developing viral

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constructs that provide chronic secretion of an RXFP3 agonist or antagonist into a local brain area (McCown, 2006; Ganella et al., 2010), or into the cerebrospinal fluid (Regev et al., 2010). This approach offer advantages over traditional chronic infusion methods (e.g. with osmotic minipumps), as surgery is performed only once, and secretion can last for months. A major ongoing question in the relaxin-3 research field pertains to the neurochemical ‘phenotype’ of RXFP3-positive neurons in different brain areas. Although the regional distribution of RXFP3 has been mapped, most positive brain regions contain numerous heterogeneous populations of neurons that have well defined connections and functions and express specific transmitters and marker proteins. Although the ‘traditional’ approach to such neuronal phenotyping has been hindered by the unavailability of suitably-sensitive RXFP3 antisera, a recently developed transgenic mouse strain (BAC-eGFP-RXFP3; Gong et al., 2003) may facilitate studies of this type, in addition to more targeted physiological studies such as recording directly from identified neurons in brain slices. Finally, further studies of human and non-human primate tissues are required to better assess the relevance of rodent anatomical and experimental data to human physiology and pathology. 12. Conclusions The first 10 years of relaxin-3 research has yielded some exciting insights. Studies investigating the neuroanatomical distribution of relaxin-3 and RXFP3 indicate that relaxin-3 containing GABAergic neurons are an integral part of an ascending arousal pathway that could potentially modulate a broad range of neural circuits and associated physiology and behaviours under different conditions, including spatial and emotional memory, the response to stress, feeding and metabolism, motivation and reward, circadian rhythm and sleep/wake states. Clearly, the majority of these putative modulatory functions need to be confirmed with further detailed physiological studies, but this is now far more feasible using an excellent range of experimental tools, including selective RXFP3 agonist/antagonist peptides, relevant transgenic mouse strains, and new viral-based approaches. Using these and other emerging technical approaches, the next decade of research promises to significantly improve our understanding of relaxin-3/RXFP3 network function, and potentially drive the development of RXFP3-based treatments for mental illnesses, including anxiety and depression.

Acknowledgments The research in the authors’ laboratory reviewed here was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia and the Pratt and Besen Foundations, a research agreement with Johnson & Johnson PR&D LLC, San Diego, USA and by the Victorian Government Strategic Investment. C.M.S. is a Florey Foundation Fellow, P.J.R. is the recipient of an Australian Postgraduate Award and a Dowd Foundation Scholarship, S.M. is an NHMRC (Australia) Australian Biomedical Fellow, and A.L.G. is an NHMRC (Australia) Senior Research Fellow. The authors wish to acknowledge their many colleagues who supported and contributed to the research reviewed in this article.

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