Sensitivity to Chronic Methamphetamine Administration and Withdrawal in Mice with Relaxin-3/RXFP3 Deficiency.

Methamphetamine (METH) is a highly addictive psychostimulant, and cessation of use is associated with reduced monoamine signalling, and increased anxiety/depressive states. Neurons expressing the neuropeptide, relaxin-3 (RLN3), and its cognate receptor, RXFP3, constitute a putative 'ascending arousal system', which shares neuroanatomical and functional similarities with serotonin (5-HT)/dorsal raphe and noradrenaline (NA)/locus coeruleus monoamine systems. In light of possible synergistic roles of RLN3 and 5-HT/NA, endogenous RLN3/RXFP3 signalling may compensate for the temporary reduction in monoamine signalling associated with chronic METH withdrawal, which could alter the profile of 'behavioural despair', bodyweight reductions, and increases in anhedonia and anxiety-like behaviours observed following chronic METH administration. In studies to test this theory, Rln3 and Rxfp3 knockout (KO) mice and their wildtype (WT) littermates were injected once daily with saline or escalating doses of METH (2 mg/kg, i.p. on day 1, 4 mg/kg, i.p. on day 2 and 6 mg/kg, i.p. on day 3-10). WT and Rln3 and Rxfp3 KO mice displayed an equivalent sensitivity to behavioural despair (Porsolt swim) during the 2-day METH withdrawal and similar bodyweight reductions on day 3 of METH treatment. Furthermore, during a 3-week period after the cessation of chronic METH exposure, Rln3 KO, Rxfp3 KO and corresponding WT mice displayed similar behavioural responses in paradigms that measured anxiety (light/dark box, elevated plus maze), anhedonia (saccharin preference), and social interaction. These findings indicate that a whole-of-life deficiency in endogenous RLN3/RXFP3 signalling does not markedly alter behavioural sensitivity to chronic METH treatment or withdrawal, but leave open the possibility of a more significant interaction with global or localised manipulations of this peptide system in the adult brain.

Involvement of central relaxin-3 signalling in sodium (salt) appetite.

NEW FINDINGS: What is the central question of this study? Sodium appetite is controlled by conserved neuronal transmitter-receptor systems. Here, we tested the contribution made by relaxin family peptide 3 receptor (RXFP3), the cognate G-protein-coupled receptor for the neuropeptide relaxin-3. What is the main finding and its importance? Intracerebroventricular infusion of an RXFP3 antagonist reduced in a dose-dependent manner the volume of 0.3 m NaCl consumed by sodium-depleted C57Bl/6J (wild-type) mice. This effect was absent in sodium-depleted Rxfp3 knockout mice, and RXFP3 antagonist infusion did not alter water consumption in wild-type mice subjected to multiple thirst tests, indicating both the pharmacological and the physiological specificity of observed effects. Our findings identify endogenous relaxin-3-RXFP3 signalling as a modulator of sodium appetite. Overconsumption of highly salted foods is common in Western diets and contributes significantly to metabolic disorders such as hypertension, renal dysfunction and diabetes. Sodium appetite, or the desire of terrestrial animals to seek and consume sodium-containing salts, is a behaviour mediated by a set of evolutionarily conserved neuronal systems. In these studies, we tested whether this instinctive behavioural drive is influenced by the G-protein-coupled relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the neuropeptide relaxin-3, because relaxin-3-RXFP3 signalling can modulate arousal, motivation and ingestive behaviours. Intracerebroventricular (i.c.v.) infusion of the selective RXFP3 antagonist, R3(B1-22)R, reduced in a dose-dependent manner the volume of 0.3 m NaCl solution consumed when offered to sodium-depleted C57Bl/6J wild-type mice, relative to vehicle-treated control animals. Notably, i.c.v. R3(B1-22)R infusion did not alter 0.3 m NaCl consumption relative to vehicle in sodium-depleted Rxfp3 knockout mice, confirming the pharmacological specificity of this effect. Furthermore, i.c.v. R3(B1-22)R did not alter the volume of water consumed by wild-type mice in three tests where water drinking was the normal physiological response, suggesting that the ability of R3(B1-22)R to reduce activated salt appetite is specific and not due to a generalized reduction in drinking behaviour. These findings identify, for the first time, that endogenous relaxin-3-RXFP3 signalling is a powerful mediator of salt appetite in mice and further elucidate the functional role of the relaxin-3-RXFP3 system in the integrative control of motivated behaviours.

Central relaxin-3 receptor (RXFP3) activation reduces elevated, but not basal, anxiety-like behaviour in C57BL/6J mice.

Anxiety disorders are among the most prevalent neuropsychiatric conditions, but their precise aetiology and underlying pathophysiological processes remain poorly understood. In light of putative anatomical and functional interactions of the relaxin-3/RXFP3 system with anxiety-related neural circuits, we assessed the ability of central administration of the RXFP3 agonist, RXFP3-A2, to alter anxiety-like behaviours in adult C57BL/6J mice. We assessed how RXFP3-A2 altered performance in tests measuring rodent anxiety-like behaviour (large open field (LOF), elevated plus maze (EPM), light/dark (L/D) box, social interaction). We examined effects of RXFP3-A2 on low 'basal' anxiety, and on elevated anxiety induced by the anxiogenic benzodiazepine, FG-7142; and explored endogenous relaxin-3/RXFP3 signalling modulation by testing effects of an RXFP3 antagonist, R3(B1-22)R, on these behaviours. Intracerebroventricular (icv) injection of RXFP3-A2 (1 nmol, 15 min pre-test) did not alter anxiety-like behaviour under 'basal' conditions in the LOF, EPM or L/D box, but reduced elevated indices of FG-7142-induced (30 mg/kg, ip) anxiety-like behaviour in the L/D box and a single-chamber social interaction test. Furthermore, R3(B1-22)R (4 nmol, icv, 15 min pre-test) increased anxiety-like behaviour in the EPM (reflected by reduced entries into the open arms), but not consistently in the LOF, L/D box or social interaction tests, suggesting endogenous signaling only weakly participates in regulating 'basal' anxiety-like behaviour, in line with previous studies of relaxin-3 and RXFP3 gene knockout mice. Overall, these data suggest exogenous RXFP3 agonists can reduce elevated (FG-7142-induced) levels of anxiety in mice; data important for gauging how conserved such effects are, with a view to modelling human pathophysiology and the likely therapeutic potential of RXFP3-targeted drugs.

Relaxin-3 receptor (RXFP3) signalling mediates stress-related alcohol preference in mice.

Stressful life events are causally linked with alcohol use disorders (AUDs), providing support for a hypothesis that alcohol consumption is aimed at stress reduction. We have previously shown that expression of relaxin-3 mRNA in rat brain correlates with alcohol intake and that central antagonism of relaxin-3 receptors (RXFP3) prevents stress-induced reinstatement of alcohol-seeking. Therefore the objectives of these studies were to investigate the impact of Rxfp3 gene deletion in C57BL/6J mice on baseline and stress-related alcohol consumption. Male wild-type (WT) and Rxfp3 knockout (KO) (C57/B6JRXFP3TM1/DGen) littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in mice following chronic alcohol intake and in naive controls. WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and hepatic alcohol processing. However, Rxfp3 KO mice displayed a stress-induced reduction in alcohol preference that was not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing effects of alcohol.

Relaxin-3/RXFP3 networks: an emerging target for the treatment of depression and other neuropsychiatric diseases?

Animal and clinical studies of gene-environment interactions have helped elucidate the mechanisms involved in the pathophysiology of several mental illnesses including anxiety, depression, and schizophrenia; and have led to the discovery of improved treatments. The study of neuropeptides and their receptors is a parallel frontier of neuropsychopharmacology research and has revealed the involvement of several peptide systems in mental illnesses and identified novel targets for their treatment. Relaxin-3 is a newly discovered neuropeptide that binds, and activates the G-protein coupled receptor, RXFP3. Existing anatomical and functional evidence suggests relaxin-3 is an arousal transmitter which is highly responsive to environmental stimuli, particularly neurogenic stressors, and in turn modulates behavioral responses to these stressors and alters key neural processes, including hippocampal theta rhythm and associated learning and memory. Here, we review published experimental data on relaxin-3/RXFP3 systems in rodents, and attempt to highlight aspects that are relevant and/or potentially translatable to the etiology and treatment of major depression and anxiety. Evidence pertinent to autism spectrum and metabolism/eating disorders, or related psychiatric conditions, is also discussed. We also nominate some key experimental studies required to better establish the therapeutic potential of this intriguing neuromodulatory signaling system, including an examination of the impact of RXFP3 agonists and antagonists on the overall activity of distinct or common neural substrates and circuitry that are identified as dysfunctional in these debilitating brain diseases.

Central injection of relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice.

Behavioural arousal in mammals is regulated by various interacting central monoamine- and peptide-neurotransmitter/receptor systems, which function to maintain awake, alert and active states required for performance of goal-directed activities essential for survival, including food seeking. Existing anatomical and functional evidence suggests the highly-conserved neuropeptide, relaxin-3, which signals via its cognate Gi/o-protein coupled receptor, RXFP3, contributes to behavioural arousal and feeding behaviour in rodents. In studies to investigate this possibility further, adult male C57BL/6J mice were treated with the selective RXFP3 antagonist peptides, R3(B1-22)R/I5(A) and R3(B1-22)R, and motivated food seeking and consumption was assessed as a reflective output of behavioural arousal. Compared to vehicle treatment, intracerebroventricular (icv) injection of RXFP3 antagonists reduced: (i) food anticipatory activity before meal time during food restriction; (ii) consumption of highly palatable food; (iii) consumption of regular chow during the initial dark phase, and; (iv) consumption of regular chow after mild (∼4-h) food deprivation. Effects were not due to sedation and appeared to be specifically mediated via antagonism of relaxin-3/RXFP3 signalling, as RXFP3 antagonist treatment did not alter locomotor activity in wild-type mice or reduce palatable food intake in relaxin-3 deficient (knock-out) mice. Notably, in contrast to similar studies in the rat, icv injection of RXFP3 agonists and infusion into the paraventricular hypothalamic nucleus did not increase food consumption in mice, suggesting species differences in relaxin-3/RXFP3-related signalling networks. Together, our data provide evidence that endogenous relaxin-3/RXFP3 signalling promotes motivated food seeking and consumption, and in light of the established biological and translational importance of other arousal systems, relaxin-3/RXFP3 networks warrant further experimental investigation.

Viral-mediated delivery of an RXFP3 agonist into brain promotes arousal in mice.

Anatomical and functional studies of central relaxin-3/RXFP3 systems suggest they constitute an ascending arousal network. For example, relaxin-3 knockout mice display circadian hypoactivity compared to wild type littermate controls. In studies to explore the effect of chronic RXFP3 activation on behaviour, we engineered a lentiviral construct to constitutively secrete the RXFP3 agonist, R3/I5, and express a green fluorescent protein (GFP) marker in transduced cells. Intracerebroventricular injection of the lenti-R3/I5-GFP virus (-10(power)8 genomic copies in 2 microl) in adult C57BL/6J mice resulted in GFP expression within cells of the ventricle walls and choroid plexus over a period of 1-4 weeks, suggesting likely chronic R3/I5 secretion and RXFP3 activation in brain regions proximal to the ventricular system. Subsequent testing in automated locomotor cells on day 8 and 9 post-injection revealed that lenti-R3/I5-GFP treated mice displayed prolonged, elevated locomotor activity (-18% higher over the last 15 min on day 8, and over the entire 30 min test on day 9) compared to mice injected with a control lenti-GFP virus, which habituated normally to the novel environment (n = 18/12 respectively, p < 0.05). These findings are consistent with an earlier report of increased activity scores in rats acutely injected centrally with R3/I5, and further suggest a role for relaxin-3/RXFP3 signalling in promoting behavioural arousal.

Consequences of relaxin-3 null mutation in mice on food-entrainable arousal.

Relaxin-3/RXFP3 networks have been hypothesised to influence behavioural state based on their anatomical distribution and recent experimental findings in rat and mouse. Two arousal-related behaviours altered by changes in relaxin-3/RXFP3 signalling are feeding and voluntary running wheel activity. In particular, relaxin-3 null mutation (knockout) mice display a 'dark-phase hypoactivity' phenotype, reflected by reduced voluntary running wheel activity and increased sleeping behaviour, with no other major changes in basal behavioural profile. The present study compared the ability of relaxin-3 deficient (null mutation) and C57BL/6J wildtype littermate mice to entrain daily running wheel activity to timed food availability. Both genotypes adjusted to a restricted feeding paradigm of 3 hours access from ZT6 to ZT9 for 14 days and displayed increased running wheel activity in the 3 hour period prior to scheduled feeding, a phenomena termed food anticipatory activity. No significant difference in running wheel activity was observed between the genotypes, indicating that a whole-of-life relaxin-3 deficiency does not prevent entrainment to a restricted-feeding schedule. Further studies of the precise interaction between relaxin-3/RXFP3 signalling and the other major arousal networks are ongoing, using currently available and new strains of transgenic mice in combination with pharmacological and viral-based methods.

Potential hypothalamic targets of relaxin-3 innervation: a perspective.

Relaxin-3 is a recently identified neuropeptide transmitter primarily expressed by neurons of the pontine nucleus incertus, which binds/activates the G(i/o)-protein coupled receptor, RXFP3. Functional studies have demonstrated that relaxin-3 modulates behavioural arousal in rodents, and although initial anatomical mapping studies have revealed relaxin-3-positive projections within several brain regions containing neurons that control behavioural arousal, further analysis of this topography has been hampered by the unavailability of a suitable specific RXFP3 antibody. In an effort to determine some of the neuron populations that relaxin-3 signalling directly modulates, we examined the distribution of relaxin-3 immunoreactive nerve fibres/terminals within the mouse lateral hypothalamus (LH) and ventrolateral preoptic area (VLPO), relative to elements containing protein markers for arousal-related neurons. Within the LH, relaxin-3 fibres were predominately distributed more laterally than orexin immunoreactive neurons, in the so-called 'parvalbumin-immunoreactive' PV1 region; but direct contacts with these parvalbumin neurons were scarce. Within the VLPO, relaxin-3 fibres were observed in close contact with galanin-immunoreactive elements, but the soma of the galanin/GABA neurons in the area that project to and inhibit arousal-promoting neurons such as orexin/LH cells to promote sleep, were not identified under the conditions employed. Nonetheless, these preliminary studies suggest an interaction between relaxin-3 and VLPO galanin neurons that may contribute to the arousal-promoting action of relaxin-3.

Pharmacological activation of RXFP3 is not orexigenic in C57BL/6J mice.

The neuropeptide relaxin-3 and its cognate G-protein-coupled receptor, RXFP3, have been implicated in the control of feeding behaviour in rats. For example, relaxin-3-positive projections and RXFP3 are present within hypothalamic feeding circuits, and icv injection of human relaxin-3 (-0.2 to 1.0 nmol) robustly increases feeding behaviour in satiated rats. To explore whether this action is conserved in other experimental species, the present study examined feeding behaviour in C57BL/6J mice following RXFP3 modulation, as mice display near identical regional distribution patterns of relaxin-3/RXFP3, and relaxin-3/RXFP3 signalling has been shown to modulate behavioural arousal in both species. Central injection of the RXFP3 agonists R3/I5 or H3 relaxin (0.5 nmol, icv) did not alter chow consumption in satiated mice relative to vehicle controls, during the 60 min after treatment. Furthermore, relaxin-3 knockout mice displayed similar basal 24-h chow consumption and 1-h palatable food consumption to wildtype littermate controls; although further studies involving acute pharmacological antagonism of RXFP3 in WT mice are required to eliminate the likelihood of compensation in these life-long relaxin-3 deficient mice. Taken together, these findings are in contrast to the potent orexigenic effects of RXFP3 activation observed in rats, and may reflect differential RXFP3 expression within hypothalamic neuron populations in the rat and mouse, or differences in signalling upstream or downstream of relaxin-3/RXFP3 networks in these two species.

Synthesis of fluorescent analogs of relaxin family peptides and their preliminary in vitro and in vivo characterization.

Relaxin, a heterodimeric polypeptide hormone, is a key regulator of collagen metabolism and multiple vascular control pathways in humans and rodents. Its actions are mediated via its cognate G-protein-coupled receptor, RXFP1 although it also "pharmacologically" activates RXFP2, the receptor for the related, insulin-like peptide 3 (INSL3), which has specific actions on reproduction and bone metabolism. Therefore, experimental tools to facilitate insights into the distinct biological actions of relaxin and INSL3 are required, particularly for studies of tissues containing both RXFP1 and RXFP2. Here, we chemically functionalized human (H2) relaxin, the RXFP1-selective relaxin analog H2:A(4-24)(F23A), and INSL3 to accommodate a fluorophore without marked reduction in binding or activation propensity. Chemical synthesis of the two chains for each peptide was followed by sequential regioselective formation of their three disulfide bonds. Click chemistry conjugation of Cy5.5 at the B-chain N-terminus, with conservation of the disulfide bonds, yielded analogs displaying appropriate selective binding affinity and ability to activate RXFP1 and/or RXFP2 in vitro. The in vivo biological activity of Cy5.5-H2 relaxin and Cy5.5-H2:A(4-24)(F23A) was confirmed in mice, as acute intracerebroventricular (icv) infusion of these peptides (but not Cy5.5-INSL3) stimulated water drinking, an established behavioral response elicited by central RXFP1 activation. The central distribution of Cy5.5-conjugated peptides was examined in mice killed 30 min after infusion, revealing higher fluorescence within brain tissue near-adjacent to the cerebral ventricle walls relative to deeper brain areas. Production of fluorophore-conjugated relaxin family peptides will facilitate future pharmacological studies to probe the function of H2 relaxin/RXFP1 and INSL3/RXFP2 signaling in vivo while tracking their distribution following central or peripheral administration.