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

Highlights

• Current treatments for anxiety disorders are inadequate.

• Relaxin-3/RXFP3 system has potential as a therapeutic target.

• Icv RXFP3 agonist did not reduce basal anxiety in mice.

• Icv RXFP3 agonist reduced pharmacologically-induced anxiety.

• Icv RXFP3 antagonist induced anxiety in the elevated plus maze test.

• Relaxin-3/RXFP3 effects on anxiety are conserved between mice and rats.

Abstract

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.

Introduction

Anxiety disorders comprise a highly prevalent, heterogeneous group of clinically defined psychiatric conditions [1], [2], which stem from a wide range of potential causes [3], [4], and are often co-morbid with other psychiatric conditions such as addiction or mood disorders [5], [6]. Elevated levels of anxiety are often associated with heightened stress, linked to combined developmental and traumatic life-events and can be modelled experimentally in animals by subjecting them to acute or chronic stressors, such as predator exposure, neonatal isolation, social defeat, restraint, or chronic unpredictable stress [7]; or treatment with anxiogenic drugs, such as yohimbine or FG-7142 [8], [9], [10]. Using these animal models, it is possible to investigate the effectiveness of putative novel treatments and identify new molecular targets or signaling systems for improving existing therapeutics [11], [12].

Relaxin-3 is a highly conserved neuropeptide [13], which acts via a single cognate G_i/o-protein-coupled receptor, relaxin family peptide receptor 3 (RXFP3) [14]. The largest population of relaxin-3 expressing neurons is located within the tegmental area known as the nucleus incertus (NI), and these neurons project broadly throughout the brain [15], [16], [17], [18], [19]. The neuroanatomy of the relaxin-3/RXFP3 system suggests a broad role as an ascending neuromodulatory network [20], [21], akin to the monoamine systems including serotonin, and noradrenaline [22], [23], [24], [25]. Anatomical and functional data [15], [16], [17], [18] suggest that relaxin-3/RXFP3 systems may interact directly with monoamine [19], [26] and other peptide systems [27], [28], [29], and/or act at shared downstream limbic and hypothalamic target areas to modulate ‘anxiety’ and other stress-related responses [30], [31], [32], [33], [34].

In rats and mice, high levels of RXFP3 mRNA are present within the amygdala, periventricular hypothalamus, ventral hippocampus, periaqueductal grey, and other brain areas/circuits [18], [35], [36] implicated in the modulation of innate anxiety and learned fear (see e.g. [6], [37], [38], [39]). Functionally, icv administration in adult Sprague–Dawley rats of the relaxin-3 agonist peptide, ‘RXFP3-A2’, which is selective for RXFP3 over RXFP1 [40], reduced anxiety-like behaviour [33]. The precise brain sites influenced by RXFP3 to produce these behavioral changes in the anxiety tests studied are not known, but it might involve activation of RXFP3 strongly expressed within areas involved in aversion-motivated exploration such as the extended amygdala, the periaqueductal grey and hippocampus [29], [41], [42], [43].

Relatively few experimental studies of the relaxin-3/RXFP3 system have been conducted in mice, but comparative studies with those in rats can indicate how functionally conserved this system is, and indicate the likelihood that findings are translatable to humans. For example, increased feeding and modest body weight gain following acute and chronic central RXFP3 activation is well characterized in the rat (see [31] for review), but icv or intra-hypothalamic injection of a specific RXFP3 agonist does not induce food intake in mice [44] and studies of relaxin-3 and RXFP3 knockout (KO) mice reveal no differences in body weight relative to their wildtype (WT) littermates [45], [46].

In this study we assessed the ability of centrally administered RXFP3 agonist to alter anxiety-like behaviour as reflected by performance in several tests - large open-field (LOF), elevated plus maze (EPM), light/dark (L/D) box and a social interaction test, which have been shown to be effective in detecting effects of new and existing anxiolytic and anxiogenic drugs [47]. We used the selective RXFP3 agonist, RXFP3-A2 [40], and assessed whether exogenous RXFP3 activation altered basal levels or ‘elevated, stress-induced’ levels of anxiety produced by administration of the partial inverse benzodiazepine agonist, FG-7142, an established anxiogenic drug (see [48]). We also examined the effects of centrally administrated R3(B1-22)R, an RXFP3 antagonist [49], on performance of mice in these same tests of anxiety-like behaviour, to determine whether endogenous relaxin-3/RXFP3 signaling modulates these outputs.