Research reportCentral injection of relaxin-3 receptor (RXFP3) antagonist peptides reduces motivated food seeking and consumption in C57BL/6J mice
Introduction
The mammalian brainstem contains several distinct neural loci which send broad ascending projections to the forebrain, and which have been well studied for their roles in arousal and related behaviours (e.g. [1], [2], [3]). These include the locus coeruleus, dorsal and median raphé nuclei, and the pedunculopontine/laterodorsal tegmental nuclei (PPT/LDTg), which contain neurons that release noradrenaline [4], serotonin (5-HT) [5] and acetylcholine [6], respectively [1], [2], [3]. A fundamental function of brainstem ascending arousal pathways is the control of alertness and sleep/wake states; but they also modulate levels of motivation, responses to stress, exploratory drive and spatial navigation, and other functions required for animals to survive, avoid predators, and acquire food (e.g. [1], [2], [3], [5]). The function of these ascending arousal pathways is complemented by other broadly projecting systems, such as orexin (hypocretin)-expressing neurons in the lateral, dorsal and perifornical hypothalamic regions [7], which have been implicated in the control of arousal/sleep, food intake and reward [1], [3], [8].
Relaxin-3 is a highly conserved neuropeptide [9], [10], [11], [12], which signals via its cognate G-protein-coupled receptor, relaxin family peptide 3 receptor (RXFP3; formerly GPCR135 [13], [14]), which also has a spatiotemporally conserved expression pattern spanning zebrafish (Danio rerio) to mammals [15], [16], [17], [18], [19] (see [20], [21], [22] for review). Existing evidence suggests neurons that express relaxin-3 constitute a newly identified ascending brainstem arousal network [22], [23]. For example, relaxin-3 neurons within the pontine nucleus incertus (NI) project broadly throughout the brain to innervate septohippocampal, hypothalamic and other limbic circuits in a regional pattern that strongly parallels noradrenaline/locus coeruleus and 5-HT/raphé systems [11], [16], [18].
Interconnectivity is a hallmark of monoamine/peptide arousal systems, and relaxin-3/NI neurons have strong reciprocal connections with the dorsal raphé nucleus and lateral hypothalamus in the rat [24], [25]. Relaxin-3/NI neurons express receptors for 5-HT [26] and orexin [27], and 5-HT depletion increased NI relaxin-3 expression in rats [26], while in a preliminary electrophysiological study, bath application of orexins activated NI relaxin-3 and non-relaxin-3 neurons [28]. Relaxin-3-positive neurons are also present within the pontine raphé nucleus, a region dorsal to the substantial nigra, and in the medial and ventrolateral periaqueductal grey in rat [11], [16] and mouse [18] brain. Although studied in less detail, initial reports suggest an ability of these populations to similarly contribute to behavioural arousal. For example, relaxin-3 neurons within the periaqueductal grey project to the thalamic intergeniculate leaflet [29], a key integration node for photic and non-photic information in diurnal and nocturnal species [30]. Application of an RXFP3 agonist was observed to stimulate neuropeptide Y neurons within the intergeniculate leaflet, which project to the suprachiasmatic nucleus to modulate circadian rhythm and arousal [30], [31], [32].
The ‘classical’ transmitter within NI neurons is GABA, reflected by the presence of glutamate decarboxylase [16], although some neurons appear to have an excitatory phenotype and express vesicular glutamate transporter [16], [33] (Allen Brain Atlas, 〈www.brain-map.org〉). The relative contribution that amino acid and peptide transmitters (especially GABA and relaxin-3) make to the overall or specific functions of these neurons is unknown. However, a variety of in vivo pharmacological studies in rodents suggest relaxin-3/RXFP3 signalling is capable of significantly modulating neuronal processes and behaviours, in a manner similar to, and perhaps synergistic with, other monoamine and peptide arousal transmitters. These studies initially examined the effect of central injection/infusion of native human relaxin-3 [34], [35]; although due to the ability of human relaxin-3 to bind/activate RXFP3 and both RXFP1 and RXFP4 in vitro and/or in vivo [36], [37], specific RXFP3 agonist and antagonist peptides have been developed to avoid potential ‘cross-reactivity’ of relaxin-3 which may not represent endogenous function, but which occurs following central injection of exogenous native peptide [38], [39], [40]. Truncation of residues from both the N- and C-terminals of the relaxin-3 A-chain and replacement of a cysteine with an arginine resulted in the formation of a specific RXFP3 agonist (referred to as ‘Analogue 2’ or RXFP3-A2 [41], and designated here as R3(B)/R3(A11-24)). The first selective RXFP3 antagonist developed consisted of a truncated relaxin-3 B-chain with an additional C-terminal arginine, conjugated to the insulin-like peptide 5 A-chain (referred to as R3(BΔ23-27)/I5 [42], designated here as R3(B1-22)R/I5(A)). It was subsequently demonstrated that with a serine replacing a cysteine, this truncated relaxin-3 B-chain alone acts as a stable and specific RXFP3 antagonist (designated R3(B1-22)R) [43].
Several behavioural studies in rats have demonstrated that pharmacological modulation of RXFP3 by central injection of these peptides can alter arousal-related activities, such as the response to stress [44], [45] and hippocampal theta rhythm [46]. In particular, effects on feeding have been well documented (see [47] for review). Intracerebroventricular (icv) injection of human relaxin-3 and specific RXFP3 agonists including R3(B)/R3(A11-24), at doses ranging from 0.18 to 1.1 nmol, increased food consumption during the hour after administration in satiated rats [34], [41]. These orexigenic effects were blocked via pre-injection of higher doses (4–4.8 nmol) of RXFP3 antagonists, including R3(B1-22)R [41], [42], [43]. Although the identity of the population(s) of RXFP3-expressing neurons responsible for mediating these acute effects is unknown, they are likely to reside within the hypothalamus, as local infusion of 0.18 nmol human relaxin-3 into the paraventricular (PVN), arcuate, and other hypothalamic nuclei also stimulates feeding [39].
Near identical neuroanatomical distributions of relaxin-3 and RXFP3 within rats and mice [16], [18] predict similar roles for this system in arousal in these species, assuming upstream inputs and downstream signalling components are also similar. In support of this theory, relaxin-3 knockout (KO) mice run less distance on voluntary, free access, home-cage running wheels compared to wildtype (WT) littermate controls during the dark/active phase [48], suggesting endogenous murine relaxin-3/RXFP3 signalling promotes arousal and motivated behaviour. In comparison to the rat, however, the impact of pharmacological modulation of RXFP3 in mice remains under explored. Such studies are important to determine whether effects are conserved across species, and can utilize transgenic mouse strains, such as relaxin-3 KO mice [48], to assess whether pharmacological effects are specifically due to the blockade of endogenous relaxin-3/RXFP3 signalling.
In studies to explore the role of endogenous murine relaxin-3/RXFP3 signalling in motivated feeding behaviours, we made icv injections of RXFP3 antagonists [34], [43] in mice and assessed: (i) food seeking behaviour in the period before meal time during food restriction [49], [50]; (ii) consumption of highly palatable food [51], and; consumption of regular chow (iii) during the light phase and initial dark phase and (iv) following ‘mild’ food deprivation. These studies revealed that acute pharmacological RXFP3 antagonism significantly reduced ‘motivated’ food seeking and consumption in mice. These effects were observed in the absence of effects on locomotor activity in automated locomotor cells, and icv RXFP3 antagonist injection did not alter the behaviour of relaxin-3 KO mice, suggesting pharmacological specificity of the peptide treatments used. Interestingly, icv injection and intra-PVN infusion of RXFP3 agonists did not increase feeding behaviour in satiated or mildly food deprived mice, indicating differences in the role of relaxin-3/RXFP3 signalling in the mouse and the rat. These data provide further evidence that endogenous relaxin-3/RXFP3 signalling drives food consumption and possibly general motivation, in line with a similar role played by other arousal signalling systems.
Section snippets
Animals
All procedures were undertaken with approval from The Florey Institute of Neuroscience and Mental Health Animal Welfare Committee, in strict compliance with the ethical guidelines of the National Health and Medical Research Council of Australia. Adult male C57BL/6J mice were obtained from the Australian Research Centre (Canning Vale, WA, Australia), while backcrossed male C57BL/6J relaxin-3 knockout (KO) and wildtype (WT) littermate mice (Lexicon Genetics Inc., The Woodlands, TX, USA) were
Icv injection of an RXFP3 antagonist reduced food anticipatory activity
Food restricted, vehicle-treated mice spent ∼36% of 60 min before meal time climbing on the cage lid (indicative of FAA and food seeking behaviour), which was significantly greater (70-fold) than the ∼0.5% of time spent climbing by free-fed vehicle controls (one-way ANOVA: post-test, P < 0.001; Fig. 2). Icv injection of the RXFP3 antagonist, R3(B1-22)R/I5(A) (0.7 nmol) markedly reduced climbing behaviour in food restricted mice by >50% c.f. vehicle-treated controls (post-test, P = 0.021).
Icv injection of an RXFP3 antagonist reduced palatable food consumption
Mice were
Discussion
The major finding of this study was that acute icv injection of RXFP3 antagonist peptides reduced motivated food seeking and consumption in adult male mice, which are interpreted as measurable ‘outputs’ of behavioural arousal. These effects were not due to general sedation, and appeared specifically associated with pharmacological blockade of RXFP3.
Treatment with the chimeric peptide, R3(B1-22)R/I5(A) [42], significantly reduced food anticipatory activity (FAA). FAA enables mice to be
Conclusions
Correct modulation of behavioural arousal in humans maintains optimal attention, cognition, motivation, sleep patterns and even mood [3], [8], [85]. Not surprisingly, disruption of transmitter and neuropeptide systems that control arousal and related modalities, such as serotonin, noradrenaline and acetylcholine, and/or orexin and CRF, has been linked to multiple psychiatric diseases, including depression [86], [87], [88]. The present studies provide further evidence that relaxin-3 is an
Acknowledgements
This research was supported by National Health and Medical Research Council (Australia) project grants 509246 and 1024885 (A.L.G.) and 508995 (J.D.W.), and by the Pratt and Besen Family Foundations (A.L.G.) and the Victorian Government Operational Infrastructure Support Program. J.D.W. and A.L.G. are NHMRC (Australia) Fellows. C.Z. is the recipient of a Bethlehem Griffiths Research Foundation Scholarship, and A.W.W. is the recipient of a University of Melbourne International Scholarship
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2022, Molecular MetabolismCitation Excerpt :Relaxin-3 has not been demonstrated to be orexigenic in mice, but both relaxin-3 and Rxfp3 knockout mice demonstrate dark phase hypoactivity (assessed via voluntary home-cage wheel running) [59,60], potentially reflecting reduced motivational drive. Whilst an RXFP3-selective antagonist reduced motivated food seeking behaviour and palatable food intake in wild-type but not Rxfp3 knockout mice, icv injection of a synthetic RXFP3-agonist, which cross reacts with RXFP4, failed to stimulate chow or palatable food intake in wild type mice [61–63]. However, our own previous attempts to increase food intake by injecting INSL5 icv also failed to produce significant results [4] contrasting with the results after intraparenchymal injection presented here.
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2021, Neuroscience and Biobehavioral ReviewsCitation Excerpt :Smith et al. (2013b), however, demonstrated no change in food intake upon icv relaxin-3 or R3/I5 application, though this was studied in male C57BL/6 J mice. There was also some inconsistency between the two food studies with RXFP3 antagonist application: while Smith et al. (2014a) reported reduced food consumption upon icv or iPVN R3(B1−22)R administration in male C57BL/6 J mice, Ryan et al. (2013b) found no R3(B1−22)R effects on deprivation-stimulated food intake in male Wistar rats. Two articles reported the effects of RXFP3 antagonism on food intake in the context of binge-eating.
Targeted viral vector transduction of relaxin-3 neurons in the rat nucleus incertus using a novel cell-type specific promoter
2020, IBRO ReportsCitation Excerpt :Social recognition is also influenced by relaxin-3 signalling, as RXFP3 agonists, delivered by viral vector-mediated expression in ventral hippocampus (Rytova et al., 2019) or intracerebroventricular infusion (Albert-Gasco et al., 2018b), reduce interactions of rats with novel conspecifics. Additional pharmacological studies targeting RXFP3 have shown effects on interrelated anxiety (Ryan et al., 2013; Zhang et al., 2015), feeding (McGowan et al., 2006; Shabanpoor et al., 2012) and motivated (Smith et al., 2014a) behaviours, most likely via actions within limbic and hypothalamic networks (Kania et al., 2017). A key role for this system in innate anxiety is also highlighted by independent relaxin-3 and RXFP3 gene knock-out mouse lines, which display a small, but consistent, decrease in anxiety behaviour (Watanabe et al., 2011; Hosken et al., 2015).
Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3
2018, Journal of Biological Chemistry
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Present address: MRC Laboratory of Molecular Biology, Cambridge, UK.