Relaxin family peptide receptor 3 (RXFP3) expressing cells in the zona incerta/lateral hypothalamus augment behavioural arousal.

Fear-related psychopathologies, such as post-traumatic stress disorder, are linked to dysfunction in neural circuits that govern fear memory and arousal. The lateral hypothalamus (LH) and zona incerta (ZI) regulate fear, but our understanding of the precise neural circuits and cell types involved remains limited. Here, we examined the role of relaxin family peptide receptor 3 (RXFP3) expressing cells in the LH/ZI in conditioned fear expression and general arousal in male RXFP3-Cre mice. We found that LH/ZI RXFP3+ (LH/ZIRXFP3) cells projected strongly to fear learning, stress, and arousal centres, notably, the periaqueductal grey, lateral habenula, and nucleus reuniens. These cells do not express hypocretin/orexin or melanin-concentrating hormone but display putative efferent connectivity with LH hypocretin/orexin+ neurons and dopaminergic A13 cells. Following Pavlovian fear conditioning, chemogenetically activating LH/ZIRXFP3 cells reduced fear expression (freezing) overall but also induced jumping behaviour and increased locomotor activity. Therefore, the decreased freezing was more likely to reflect enhanced arousal rather than reduced fear. Indeed, stimulating these cells produced distinct patterns of coactivation between several motor, stress, and arousal regions, as measured by Fos expression. These results suggest that activating LH/ZIRXFP3 cells generates brain-wide activation patterns that augment behavioural arousal.

Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.

Involvement of relaxin-family peptide-3 receptor (RXFP3) in the ventral dentate gyrus of the hippocampus in spatial and fear memory in rats.

The neuropeptide relaxin-3 and its cognate receptor, relaxin family peptide-3 receptors (RXFP3), have been implicated in modulating learning and memory processes, but their specific roles remain unclear. This study utilized behavioral and molecular approaches to investigate the effects of putatively reversible blockade of RXFP3 in the ventral dentate gyrus (vDG) of the hippocampus on spatial and fear memory formation in rats. Male Wistar rats received bilateral vDG cannula implantation and injections of the RXFP3 antagonist, R3(BΔ23-27)R/I5 (400 ng/0.5 µL per side), or vehicle at specific time points before acquisition, consolidation, or retrieval phases of the Morris water maze and passive avoidance learning tasks. RXFP3 inhibition impaired acquisition in the passive avoidance task but not the spatial learning task. However, both memory consolidation and retrieval were disrupted in both tasks following RXFP3 antagonism. Ventral hippocampal levels of the consolidation-related kinase p70-S6 kinase (p70S6K) were reduced RXFP3 blockade. These findings highlight a key role for ventral hippocampal RXFP3 signaling in the acquisition, consolidation, and retrieval of spatial and emotional memories, extending previous work implicating this neuropeptide system in hippocampal memory processing.

Development of a synthetic relaxin-3/INSL5 chimeric peptide ligand for NanoBiT complementation binding assays.

INSL5 and relaxin-3 are relaxin family peptides with important roles in gut and brain function, respectively. They mediate their actions through the class A GPCRs RXFP4 and RXFP3. RXFP4 has been proposed to be a therapeutic target for colon motility disorders whereas RXFP3 targeting could be effective for neurological conditions such as anxiety. Validation of these targets has been limited by the lack of specific ligands and the availability of robust ligand-binding assays for their development. In this study, we have utilized NanoBiT complementation to develop a SmBiT-conjugated tracer for use with LgBiT-fused RXFP3 and RXFP4. The low affinity between LgBiT:SmBiT should result in a low non-specific luminescence signal and enable the quantification of binding without the tedious separation of non-bound ligands. We used solid-phase peptide synthesis to produce a SmBiT-labelled RXFP3/4 agonist, R3/I5, where SmBiT was conjugated to the B-chain N-terminus via a PEG12 linker. Both SmBiT-R3/I5 and R3/I5 were synthesized and purified in high purity and yield. Stable HEK293T cell lines expressing LgBiT-RXFP3 and LgBiT-RXFP4 were produced and demonstrated normal signaling in response to the synthetic R3/I5 peptide. Binding was first characterized in whole-cell binding kinetic assays validating that the SmBiT-R3/I5 bound to both cell lines with nanomolar affinity with minimal non-specific binding without bound and free SmBiT-R3/I5 separation. We then optimized membrane binding assays, demonstrating easy and robust analysis of both saturation and competition binding from frozen membranes. These assays therefore provide an appropriate rigorous binding assay for the high-throughput analysis of RXFP3 and RXFP4 ligands.

Developing insulin-like peptide 5-based antagonists for the G protein-coupled receptor, RXFP4.

Human insulin-like peptide 5 (INSL5) is a gut hormone produced by colonic L-cells, and its biological functions are mediated by Relaxin Family Peptide Receptor 4 (RXFP4). Our preliminary data indicated that RXFP4 agonists are potential drug leads for the treatment of constipation. More recently, we designed and developed a novel RXFP4 antagonist, A13-nR that was shown to block agonist-induced activity in cells and animal models. We showed that A13-nR was able to block agonist-induced increases in colon motility in mice of both genders that express the receptor, RXFP4. Our data also showed that colorectal propulsion induced by intracolonic administration of short-chain fatty acids was antagonized by A13-nR. Therefore, A13-nR is an important research tool and potential drug lead for the treatment of colon motility disorders, such as bacterial diarrhea. However, A13-nR acted as a partial agonist at high concentrations in vitro and demonstrated modest antagonist potency (∼35 nM). Consequently, the primary objective of this study is to pinpoint novel modifications to A13-nR that eliminate partial agonist effects while preserving or augmenting antagonist potency. In this work, we detail the creation of a series of A13-nR-modified analogues, among which analogues 3, 4, and 6 demonstrated significantly improved RXFP4 affinity (∼3 nM) with reduced partial agonist activity, enhanced antagonist potency (∼10 nM) and maximum agonist inhibition (∼80 %) when compared with A13-nR. These compounds have potential as candidates for further preclinical evaluations, marking a significant stride toward innovative therapeutics for colon motility disorders.

Targeting the relaxin-3/RXFP3 system: a patent review for the last two decades.

INTRODUCTION: The neuropeptide relaxin-3/RXFP3 system belongs to the relaxin/insulin superfamily and is involved in many important physiological processes, such as stress responses, appetite control, and motivation for reward. Although relaxin-3 is the endogenous agonist for RXFP3, it can also bind to and activate RXFP1 and RXFP4. Consequently, research has been focused on the development of RXFP3-specific peptides and small-molecule ligands to validate the relaxin-3/RXFP3 system as a novel drug target. AREAS COVERED: This review provides an overview of patents on the relaxin-3/RXFP3 system covering ligand development and pharmacological studies since 2003. Related patents and literature reports were obtained from established sources including SciFinder, Google Patents, and Espacenet for patents and SciFinder, PubMed, and Google Scholar for literature reports. EXPERT OPINION: There has been an increasing amount of patent activities around relaxin-3/RXFP3, highlighting the importance of this novel neuropeptide system for drug discovery. The development of relaxin-3 derived peptides and small-molecule modulators, as well as behavioral studies in rodents, have shown that the relaxin-3/RXFP3 system is a promising drug target for treating various metabolic and neuropsychiatric diseases including obesity, anxiety, and alcohol addiction.

Novel RXFP3 negative allosteric modulator RLX-33 reduces alcohol self-administration in rats.

There is much interest in identifying novel pharmacotherapeutic targets that improve clinical outcomes for the treatment of alcohol use disorder (AUD). One promising target for therapeutic intervention is the relaxin family peptide 3 (RXFP3) receptor, a cognate receptor for neuropeptide relaxin-3, which has previously been implicated in regulating alcohol drinking behavior. Recently, we developed the first small-molecule RXFP3-selective negative allosteric modulator (NAM) RLX-33. Therefore, the goal of the present work was to characterize the impact of this novel NAM on affective-related behaviors and alcohol self-administration in rats. First, the effects of RLX-33 were tested on alcohol and sucrose self-administration in Wistar and alcohol-preferring P rats to determine the dose-response profile and specificity for alcohol. Then, we assessed the effects of systemic RLX-33 injection in Wistar rats in a battery of behavioral assays (open-field test, elevated zero maze, acoustic startle response test, and prepulse inhibition) and tested for alcohol clearance. We found that the lowest effective dose (5 mg/kg) reduced alcohol self-administration in both male and female Wistar rats, while in alcohol-preferring P rats, this effect was restricted to males, and there were no effects on sucrose self-administration or general locomotor activity. The characterization of affective and metabolic effects in Wistar rats generally found few locomotor, affective, or alcohol clearance changes, particularly at the 5 mg/kg dose. Overall, these findings are promising and suggest that RXFP3 NAM has potential as a pharmacological target for treating AUD.

Nucleus incertus projections to rat medial septum and entorhinal cortex: rare collateralization and septal-gating of temporal lobe theta rhythm activity.

Nucleus incertus (NI) neurons in the pontine tegmentum give rise to ascending forebrain projections and express the neuropeptide relaxin-3 (RLN3) which acts via the relaxin-family peptide 3 receptor (RXFP3). Activity in the hippocampus and entorhinal cortex can be driven from the medial septum (MS), and the NI projects to all these centers, where a prominent pattern of activity is theta rhythm, which is related to spatial memory processing. Therefore, we examined the degree of collateralization of NI projections to the MS and the medial temporal lobe (MTL), comprising medial and lateral entorhinal cortex (MEnt, LEnt) and dentate gyrus (DG), and the ability of the MS to drive entorhinal theta in the adult rat. We injected fluorogold and cholera toxin-B into the MS septum and either MEnt, LEnt or DG, to determine the percentage of retrogradely labeled neurons in the NI projecting to both or single targets, and the relative proportion of these neurons that were RLN3-positive ( +). The projection to the MS was threefold stronger than that to the MTL. Moreover, a majority of NI neurons projected independently to either MS or the MTL. However, RLN3 + neurons collateralize significantly more than RLN3-negative (-) neurons. In in vivo studies, electrical stimulation of the NI induced theta activity in the MS and the entorhinal cortex, which was impaired by intraseptal infusion of an RXFP3 antagonist, R3(BΔ23-27)R/I5, particularly at ~ 20 min post-injection. These findings suggest that the MS plays an important relay function in the NI-induced generation of theta within the entorhinal cortex.

Postnatal development of the relaxin-3 innervation of the rat medial septum.

Introduction: The septal area provides a rich innervation to the hippocampus regulating hippocampal excitability to different behavioral states and modulating theta rhythmogenesis. However, little is known about the neurodevelopmental consequences of its alterations during postnatal development. The activity of the septohippocampal system is driven and/or modulated by ascending inputs, including those arising from the nucleus incertus (NI), many of which contain the neuropeptide, relaxin-3 (RLN3). Methods: We examined at the molecular and cellular level the ontogeny of RLN3 innervation of the septal area in postnatal rat brains. Results: Up until P13-15 there were only scattered fibers in the septal area, but a dense plexus had appeared by P17 that was extended and consolidated throughout the septal complex by P20. There was a decrease in the level of colocalization of RLN3 and synaptophysin between P15 and P20 that was reversed between P20 and adulthood. Biotinylated 3-kD dextran amine injections into the septum, revealed retrograde labeling present in the brainstem at P10-P13, but a decrease in anterograde fibers in the NI between P10-20. Simultaneously, a differentiation process began during P10-17, resulting in fewer NI neurons double-labeled for serotonin and RLN3. Discussion: The onset of the RLN3 innervation of the septum complex between P17-20 is correlated with the onset of hippocampal theta rhythm and several learning processes associated with hippocampal function. Together, these data highlight the relevance and need for further analysis of this stage for normal and pathological septohippocampal development.

Characterization of relaxin 3 and its receptors in chicken: Evidence for relaxin 3 acting as a novel pituitary hormone.

Mammalian relaxin (RLN) family peptides binding their receptors (RXFPs) play a variety of roles in many physiological processes, such as reproduction, stress, appetite regulation, and energy balance. In birds, although two relaxin family peptides (RLN3 and INSL5) and four receptors (RXFP1, RXFP2, RXFP2-like, and RXFP3) were predicated, their sequence features, signal properties, tissue distribution, and physiological functions remain largely unknown. In this study, using chickens as the experimental model, we cloned the cDNA of the cRLN3 gene and two receptor (cRXFP1 and cRXFP3) genes. Using cell-based luciferase reporter assays, we demonstrate that cRLN3 is able to activate both cRXFP1 and cRXFP3 for downstream signaling. cRXFP1, rather than cRXFP3, is a cognate receptor for cRLN3, which is different from the mammals. Tissue distribution analyses reveal that cRLN3 is highly expressed in the pituitary with lower abundance in the hypothalamus and ovary of female chicken, together with the detection that cRLN3 co-localizes with pituitary hormone genes LHB/FSHB/GRP/CART and its expression is tightly regulated by hypothalamic factors (GnRH and CRH) and sex steroid hormone (E2). The present study supports that cRLN3 may function as a novel pituitary hormone involving female reproduction.

High-Throughput Screening Campaign Identified a Potential Small Molecule RXFP3/4 Agonist.

Relaxin/insulin-like family peptide receptor 3 (RXFP3) belongs to class A G protein-coupled receptor family. RXFP3 and its endogenous ligand relaxin-3 are mainly expressed in the brain with important roles in the regulation of appetite, energy metabolism, endocrine homeostasis and emotional processing. It is therefore implicated as a potential target for treatment of various central nervous system diseases. Since selective agonists of RXFP3 are restricted to relaxin-3 and its analogs, we conducted a high-throughput screening campaign against 32,021 synthetic and natural product-derived compounds using a cyclic adenosine monophosphate (cAMP) measurement-based method. Only one compound, WNN0109-C011, was identified following primary screening, secondary screening and dose-response studies. Although displayed agonistic effect in cells overexpressing the human RXFP3, it also showed cross-reactivity with the human RXFP4. This hit compound may provide not only a chemical probe to investigate the function of RXFP3/4, but also a novel scaffold for the development of RXFP3/4 agonists.

The putative role of the relaxin-3/RXFP3 system in clinical depression and anxiety: A systematic literature review.

The relaxin-3/RXFP3 system is one of several neuropeptidergic systems putatively implicated in regulating the behavioural alterations that characterise clinical depression and anxiety, making it a potential target for clinical translation. Accordingly, this systematic review identified published reports on the role of relaxin-3/RXFP3 signalling in these neuropsychiatric disorders and their behavioural endophenotypes, evaluating evidence from animal and human studies to ascertain any relationship. We searched PubMed, EMBASE, PsycINFO and Google Scholar databases up to February 2021, finding 609 relevant records. After stringent screening, 51 of these studies were included in the final synthesis. There was considerable heterogeneity in study designs and some inconsistency across study outcomes. However, experimental evidence is consistent with an ability of relaxin-3/RXFP3 signalling to promote arousal and suppress depressive- and anxiety-like behaviour. Moreover, meta-analyses of six to eight articles investigating food intake revealed that acute RXFP3 activation had strong orexigenic effects in rats. This appraisal also identified the lack of high-quality clinical studies pertinent to the relaxin-3/RXFP3 system, a gap that future research should attempt to bridge.

Involvement of the Nucleus Incertus and Relaxin-3/RXFP3 Signaling System in Explicit and Implicit Memory.

Telencephalic cognitive and emotional circuits/functions are strongly modulated by subcortical inputs. The main focus of past research on the nature of this modulation has been on the widespread monoamine projections to the telencephalon. However, the nucleus incertus (NI) of the pontine tegmentum provides a strong GABAergic and peptidergic innervation of the hippocampus, basal forebrain, amygdala, prefrontal cortex, and related regions; and represents a parallel source of ascending modulation of cognitive and emotional domains. NI GABAergic neurons express multiple peptides, including neuromedin-B, cholecystokinin, and relaxin-3, and receptors for stress and arousal transmitters, including corticotrophin-releasing factor and orexins/hypocretins. A functional relationship exists between NI neurons and their associated peptides, relaxin-3 and neuromedin-B, and hippocampal theta rhythm, which in turn, has a key role in the acquisition and extinction of declarative and emotional memories. Furthermore, RXFP3, the cognate receptor for relaxin-3, is a Gi/o protein-coupled receptor, and its activation inhibits the cellular accumulation of cAMP and induces phosphorylation of ERK, processes associated with memory formation in the hippocampus and amygdala. Therefore, this review summarizes the role of NI transmitter systems in relaying stress- and arousal-related signals to the higher neural circuits and processes associated with memory formation and retrieval.

Exploring the Use of Helicogenic Amino Acids for Optimising Single Chain Relaxin-3 Peptide Agonists.

Relaxin-3 is a highly conserved two-chain neuropeptide that acts through its endogenous receptor the Relaxin Family Peptide-3 (RXFP3) receptor. The ligand/receptor system is known to modulate several physiological processes, with changes in food intake and anxiety-levels the most well studied in rodent models. Agonist and antagonist analogues based on the native two-chain peptide are costly to synthesise and not ideal drug leads. Since RXFP3 interacting residues are found in the relaxin B-chain only, this has been the focus of analogue development. The B-chain is unstructured without the A-chain support, but in single-chain variants structure can be induced by dicarba-based helical stapling strategies. Here we investigated whether alternative helical inducing strategies also can enhance structure and activity at RXFP3. Combinations of the helix inducing α-aminoisobutyric acid (Aib) were incorporated into the sequence of the relaxin-3 B-chain. Aib residues at positions 13, 17 and 18 partially reintroduce helicity and activity of the relaxin-3 B-chain, but other positions are generally not suited for modifications. We identify Thr21 as a putative new receptor contact residue important for RXFP3 binding. Cysteine residues were also incorporated into the sequence and cross-linked with dichloroacetone or α, α'-dibromo-m-xylene. However, in contrast to previously reported dicarba variants, neither were found to promote structure and RXFP3 activity.

Hydrophobic interactions of relaxin family peptide receptor 3 with ligands identified using a NanoBiT-based binding assay.

Relaxin family peptide receptor 3 (RXFP3) is a G protein-coupled receptor implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. In recent studies, interactions of RXFP3 with some natural or synthetic ligands have been investigated. In the present study, we identified the hydrophobic interactions of human RXFP3 with the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 using a newly developed NanoBiT-based homogenous binding assay. We first demonstrated that the conserved large aliphatic B15Ile and B19Ile were important for the binding of the agonist and antagonist to RXFP3, because alanine replacement significantly decreased their receptor-binding potency. Thereafter, we demonstrated that the conserved large aliphatic Leu246 and Leu248 in extracellular loop 2 were important for RXFP3 binding to the agonist and antagonist, because alanine replacement significantly decreased the binding affinity of RXFP3 for both ligands. Finally, we deduced probable hydrophobic interactions based on the ability of RXFP3 mutants to distinguish the wild-type and mutant ligands: Leu246 of RXFP3 interacted with B15Ile of both ligands, while Leu248 of RXFP3 interacted with both B15Ile and B19Ile of the agonist and antagonist. The present results not only provided new insights into the interaction mechanism of RXFP3 with agonists and antagonists, but also demonstrated usefulness of the NanoBiT-based homogenous binding assay to study the interaction mechanism of certain receptors with their ligands.

RLN3/RXFP3 Signaling in the PVN Inhibits Magnocellular Neurons via M-like Current Activation and Contributes to Binge Eating Behavior.

Binge-eating disorder is the most common eating disorder. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3 (RLN3), which stimulates food intake in rats through the activation of the relaxin-family peptide-3 receptor (RXFP3). Here we demonstrate that a likely mechanism underlying the orexigenic action of RLN3 is RXFP3-mediated inhibition of oxytocin- and arginine-vasopressin-synthesizing paraventricular nucleus (PVN) magnocellular neurosecretory cells. Moreover, we reveal that, in male and female rats, this action depends on M-like potassium conductance. Notably, higher intra- and peri-PVN RLN3 fiber densities were observed in females, which may constitute an anatomic substrate for observed sex differences in binge-eating disorder. Finally, in a model of binge-eating in female rats, RXFP3 blockade within the PVN prevented binge-eating behavior. These data demonstrate a direct RLN3/RXFP3 action in the PVN of male and female rats, identify the associated ionic mechanisms, and reveal that hypothalamic RLN3/RXFP3 signaling regulates binge-eating behavior.SIGNIFICANCE STATEMENT Binge-eating disorder is the most common eating disorder worldwide, affecting women twice as frequently as men. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3, which acts via the relaxin-family peptide-3 receptor (RXFP3). Using a model of binge-eating, we demonstrated that relaxin-3/RXFP3 signaling in the hypothalamic paraventricular nucleus (PVN) is necessary for the expression of binge-eating behavior in female rats. Moreover, we elucidated the neuronal mechanism of RLN3/RXFP3 signaling in PVN in male and female rats and characterized sex differences in the RLN3 innervation of the PVN. These findings increase our understanding of the brain circuits and neurotransmitters involved in binge-eating disorder pathology and identify RXFP3 as a therapeutic target for binge-like eating disorders.

Development of Relaxin-3 Agonists and Antagonists Based on Grafted Disulfide-Stabilized Scaffolds.

Relaxin-3 is a neuropeptide with important roles in metabolism, arousal, learning and memory. Its cognate receptor is the relaxin family peptide-3 (RXFP3) receptor. Relaxin-3 agonist and antagonist analogs have been shown to be able to modulate food intake in rodent models. The relaxin-3 B-chain is sufficient for receptor interactions, however, in the absence of a structural support, linear relaxin-3 B-chain analogs are rapidly degraded and thus unsuitable as drug leads. In this study, two different disulfide-stabilized scaffolds were used for grafting of important relaxin-3 B-chain residues to improve structure and stability. The use of both Veronica hederifolia Trypsin inhibitor (VhTI) and apamin grafting resulted in agonist and antagonist analogs with improved helicity. VhTI grafted peptides showed poor binding and low potency at RXFP3, on the other hand, apamin variants retained significant activity. These variants also showed improved half-life in serum from ~5 min to >6 h, and thus are promising RXFP3 specific pharmacological tools and drug leads for neuropharmacological diseases.

Differential Level of RXFP3 Expression in Dopaminergic Neurons Within the Arcuate Nucleus, Dorsomedial Hypothalamus and Ventral Tegmental Area of RXFP3-Cre/tdTomato Mice.

RXFP3 (relaxin-family peptide 3 receptor) is the cognate G-protein-coupled receptor for the neuropeptide, relaxin-3. RXFP3 is expressed widely throughout the brain, including the hypothalamus, where it has been shown to modulate feeding behavior and neuroendocrine activity in rodents. In order to better characterize its potential mechanisms of action, this study determined whether RXFP3 is expressed by dopaminergic neurons within the arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), in addition to the ventral tegmental area (VTA). Neurons that express RXFP3 were visualized in coronal brain sections from RXFP3-Cre/tdTomato mice, which express the tdTomato fluorophore within RXFP3-positive cells, and dopaminergic neurons in these areas were visualized by simultaneous immunohistochemical detection of tyrosine hydroxylase-immunoreactivity (TH-IR). Approximately 20% of ARC neurons containing TH-IR coexpressed tdTomato fluorescence, suggesting that RXFP3 can influence the dopamine pathway from the ARC to the pituitary gland that controls prolactin release. The ability of prolactin to reduce leptin sensitivity and increase food consumption therefore represents a potential mechanism by which RXFP3 activation influences feeding. A similar proportion of DMH neurons containing TH-IR expressed RXFP3-related tdTomato fluorescence, consistent with a possible RXFP3-mediated regulation of stress and neuroendocrine circuits. In contrast, RXFP3 was barely detected within the VTA. TdTomato signal was absent from the ARC and DMH in sections from Rosa26-tdTomato mice, suggesting that the cells identified in RXFP3-Cre/tdTomato mice expressed authentic RXFP3-related tdTomato fluorescence. Together, these findings identify potential hypothalamic mechanisms through which RXFP3 influences neuroendocrine control of metabolism, and further highlight the therapeutic potential of targeting RXFP3 in feeding-related disorders.

Characterization of the relaxin family peptide receptor 3 system in the mouse bed nucleus of the stria terminalis.

The bed nucleus of the stria terminalis (BNST) is a critical node involved in stress and reward-related behaviors. Relaxin family peptide receptor 3 (RXFP3) signaling in the BNST has been implicated in stress-induced alcohol seeking behavior. However, the neurochemical phenotype and connectivity of BNST RXFP3-expressing (RXFP3+) cells have yet to be elucidated. We interrogated the molecular signature and electrophysiological properties of BNST RXFP3+ neurons using a RXFP3-Cre reporter mouse line. BNST RXFP3+ cells are circumscribed to the dorsal BNST (dBNST) and are neurochemically heterogeneous, comprising a mix of inhibitory and excitatory neurons. Immunohistochemistry revealed that ~48% of BNST RXFP3+ neurons are GABAergic, and a quarter of these co-express the calcium-binding protein, calbindin. A subset of BNST RXFP3+ cells (~41%) co-express CaMKIIα, suggesting this subpopulation of BNST RXFP3+ neurons are excitatory. Corroborating this, RNAscope® revealed that ~35% of BNST RXFP3+ cells express vVGluT2 mRNA, indicating a subpopulation of RXFP3+ neurons are glutamatergic. RXFP3+ neurons show direct hyperpolarization to bath application of a selective RXFP3 agonist, RXFP3-A2, while around 50% of cells were depolarised by exogenous corticotrophin releasing factor. In behaviorally naive mice the majority of RXFP3+ neurons were Type II cells exhibiting Ih and T type calcium mediated currents. However, chronic swim stress caused persistent plasticity, decreasing the proportion of neurons that express these channels. These studies are the first to characterize the BNST RXFP3 system in mouse and lay the foundation for future functional studies appraising the role of the murine BNST RXFP3 system in more complex behaviors.

Septal GABA and Glutamate Neurons Express RXFP3 mRNA and Depletion of Septal RXFP3 Impaired Spatial Search Strategy and Long-Term Reference Memory in Adult Mice.

Relaxin-3 is a highly conserved neuropeptide abundantly expressed in neurons of the nucleus incertus (NI), which project to nodes of the septohippocampal system (SHS) including the medial septum/diagonal band of Broca (MS/DB) and dorsal hippocampus, as well as to limbic circuits. High densities of the Gi/o-protein-coupled receptor for relaxin-3, known as relaxin-family peptide-3 receptor (RXFP3) are expressed throughout the SHS, further suggesting a role for relaxin-3/RXFP3 signaling in modulating learning and memory processes that occur within these networks. Therefore, this study sought to gain further anatomical and functional insights into relaxin-3/RXFP3 signaling in the mouse MS/DB. Using Cre/LoxP recombination methods, we assessed locomotion, exploratory behavior, and spatial learning and long-term reference memory in adult C57BL/6J Rxfp3 loxP/loxP mice with targeted depletion of Rxfp3 in the MS/DB. Following prior injection of an AAV(1/2)-Cre-IRES-eGFP vector into the MS/DB to delete/deplete Rxfp3 mRNA/RXFP3 protein, mice tested in a Morris water maze (MWM) displayed an impairment in allocentric spatial learning during acquisition, as well as an impairment in long-term reference memory on probe day. However, RXFP3-depleted and control mice displayed similar motor activity in a locomotor cell and exploratory behavior in a large open-field (LOF) test. A quantitative characterization using multiplex, fluorescent in situ hybridization (ISH) identified a high level of co-localization of Rxfp3 mRNA and vesicular GABA transporter (vGAT) mRNA in MS and DB neurons (~87% and ~95% co-expression, respectively). Rxfp3 mRNA was also detected, to a correspondingly lesser extent, in vesicular glutamate transporter 2 (vGlut2) mRNA-containing neurons in MS and DB (~13% and ~5% co-expression, respectively). Similarly, a qualitative assessment of the MS/DB region, identified Rxfp3 mRNA in neurons that expressed parvalbumin (PV) mRNA (reflecting hippocampally-projecting GABA neurons), whereas choline acetyltransferase mRNA-positive (acetylcholine) neurons lacked Rxfp3 mRNA. These data are consistent with a qualitative immunohistochemical analysis that revealed relaxin-3-immunoreactive nerve fibers in close apposition with PV-immunoreactive neurons in the MS/DB. Together these studies suggest relaxin-3/RXFP3 signaling in the MS/DB plays a role in modulating specific learning and long-term memory associated behaviors in adult mice via effects on GABAergic neuron populations known for their involvement in modulating hippocampal theta rhythm and associated cognitive processes.