Modulation of contextual fear acquisition and extinction by acute and chronic relaxin-3 receptor (RXFP3) activation in the rat retrosplenial cortex.

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process.

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.

Some key parameters in contextual fear conditioning and extinction in adult rats.

Contextual fear conditioning is a behavioral paradigm used to assess hippocampal-dependent memory in experimental animals. Perception of the context depends on activation of a distinct population of neurons in the hippocampus and in hippocampal-related areas that process discrete aspects of context perception. In the absence of any putatively associated cue, the context becomes the salient element that may warn of an upcoming aversive event; and in particular conditions, animals generalize this warning to any new or similar context. In this study we evaluated the effects of the number of sessions, the number of unconditioned stimuli per acquisition session and the distribution of extinction sessions to assess fear acquisition and extinction and determine under which conditions generalization occurred in adult, male rats. We observed that the organization and spacing of sessions were relevant factors in the acquisition and extinction of contextual fear memories. Extinction occurred with significantly greater robustness when sessions were spread over two days. Furthermore, results indicated that exposure to a single 0.3 mA, 0.5 s footshock in two different sessions could produce context-specific fear, while more acquisition sessions or more footshocks within a single session produced a generalization of the fear response to a new context. Notably, when generalization occurred, successive re-exposure to the generalized context produced extinction in a similar way to the paired exposure. Together, the present findings identify clear procedural and behavioral parameters amenable to neural systems analysis of three clinically relevant outcomes of contextual fear conditioning, i.e., memory acquisition, storage and extinction.

CaMKK2 as an emerging treatment target for bipolar disorder.

Current pharmacological treatments for bipolar disorder are inadequate and based on serendipitously discovered drugs often with limited efficacy, burdensome side-effects, and unclear mechanisms of action. Advances in drug development for the treatment of bipolar disorder remain incremental and have come largely from repurposing drugs used for other psychiatric conditions, a strategy that has failed to find truly revolutionary therapies, as it does not target the mood instability that characterises the condition. The lack of therapeutic innovation in the bipolar disorder field is largely due to a poor understanding of the underlying disease mechanisms and the consequent absence of validated drug targets. A compelling new treatment target is the Ca2+-calmodulin dependent protein kinase kinase-2 (CaMKK2) enzyme. CaMKK2 is highly enriched in brain neurons and regulates energy metabolism and neuronal processes that underpin higher order functions such as long-term memory, mood, and other affective functions. Loss-of-function polymorphisms and a rare missense mutation in human CAMKK2 are associated with bipolar disorder, and genetic deletion of Camkk2 in mice causes bipolar-like behaviours similar to those in patients. Furthermore, these behaviours are ameliorated by lithium, which increases CaMKK2 activity. In this review, we discuss multiple convergent lines of evidence that support targeting of CaMKK2 as a new treatment strategy for bipolar disorder.

Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

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.

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.

History of key regulatory peptide systems and perspectives for future research.

Throughout the 20th Century, regulatory peptide discovery advanced from the identification of gut hormones to the extraction and characterization of hypothalamic hypophysiotropic factors, and to the isolation and cloning of multiple brain neuropeptides. These discoveries were followed by the discovery of G-protein-coupled and other membrane receptors for these peptides. Subsequently, the systems physiology associated with some of these multiple regulatory peptides and receptors has been comprehensively elucidated and has led to improved therapeutics and diagnostics and their approval by the US Food and Drug Administration. In light of this wealth of information and further potential, it is truly a time of renaissance for regulatory peptides. In this perspective, we review what we have learned from the pioneers in exemplified fields of gut peptides, such as cholecystokinin, enterochromaffin-like-cell peptides, and glucagon, from the trailblazing studies on the key stress hormone, corticotropin-releasing factor, as well as from more recently characterized relaxin-family peptides and receptors. The historical viewpoints are based on our understanding of these topics in light of the earliest phases of research and on subsequent studies and the evolution of knowledge, aiming to sharpen our vision of the current state-of-the-art and those studies that should be prioritized in the future.

Relaxin ligand/receptor systems in the developing teleost fish brain: Conserved features with mammals and a platform to address neuropeptide system functions.

The relaxins (RLNs) are a group of peptide hormone/neuromodulators that can regulate a wide range of physiological processes ranging from reproduction to brain function. All the family members have originated from a RLN3-like ancestor via different rounds of whole genome and gene specific duplications during vertebrate evolution. In mammals, including human, the divergence of the different family members and the emergence of new members led to the acquisition of specific functions for the various relaxin family peptide and associated receptor genes. In particular, in mammals, it was shown, that the role of RLN3 is correlated to the modulation of arousal, stress responses, emotion, social recognition, and other brain functions, positioning this gene/peptide as a potential therapeutic target for neuropsychiatric disorders. This review highlights the evolutionary conservation of relaxin family peptide and receptor gene expression and their associated brain neural circuits. In the zebrafish, the expression pattern of the different relaxin family members has specific features that are conserved in higher species, including a likely similar functional role for the ancestral RLN3-like gene. The use of different model organisms, particularly the zebrafish, to explore the diversification and conservation of relaxin family ligands and receptor systems, provides a relatively high-throughput platform to identify their specific conserved or differential neuromodulatory roles in higher species including human.

Catecholaminergic innervation and D2-like dopamine receptor-mediated modulation of brainstem nucleus incertus neurons in the rat.

Nucleus incertus (NI) is a brainstem structure involved in the control of arousal, stress responses and locomotor activity. It was reported recently that NI neurons express the dopamine type 2 (D2) receptor that belongs to the D2-like receptor (D2R) family, and that D2R activation in the NI decreased locomotor activity. In this study, using multiplex in situ hybridization, we observed that GABAergic and glutamatergic NI neurons express D2 receptor mRNA, and that D2 receptor mRNA-positive neurons belong to partially overlapping relaxin-3- and cholecystokinin-positive NI neuronal populations. Our immunohistochemical and viral-based retrograde tract-tracing studies revealed a dense innervation of the NI area by fibers containing the catecholaminergic biosynthesis enzymes, tyrosine hydroxylase (TH) and dopamine ß-hydroxylase (DBH), and indicated the major sources of the catecholaminergic innervation of the NI as the Darkschewitsch, raphe and hypothalamic A13 nuclei. Furthermore, using whole-cell patch clamp recordings, we demonstrated that D2R activation by quinpirole produced excitatory and inhibitory influences on neuronal activity in the NI, and that both effects were postsynaptic in nature. Moreover, the observed effects were cell-type specific, as type I NI neurons were either excited or inhibited, whereas type II NI neurons were mainly excited by D2R activation. Our results reveal that rat NI receives a strong catecholaminergic innervation and suggest that catecholamines acting within the NI are involved in the control of diverse processes, including locomotor activity, social interaction and nociceptive signaling. Our data also strengthen the hypothesis that the NI acts as a hub integrating arousal-related neuronal information.

Functional Neuroanatomy of the Rat Nucleus Incertus-Medial Septum Tract: Implications for the Cell-Specific Control of the Septohippocampal Pathway.

The medial septum (MS) is critically involved in theta rhythmogenesis and control of the hippocampal network, with which it is reciprocally connected. MS activity is influenced by brainstem structures, including the stress-sensitive, nucleus incertus (NI), the main source of the neuropeptide relaxin-3 (RLN3). In the current study, we conducted a comprehensive neurochemical and electrophysiological characterization of NI neurons innervating the MS in the rat, by employing classical and viral-based neural tract-tracing and electrophysiological approaches, and multiplex fluorescent in situ hybridization. We confirmed earlier reports that the MS is innervated by RLN3 NI neurons and documented putative glutamatergic (vGlut2 mRNA-expressing) neurons as a relevant NI neuronal population within the NI-MS tract. Moreover, we observed that NI neurons innervating MS can display a dual phenotype for GABAergic and glutamatergic neurotransmission, and that 40% of MS-projecting NI neurons express the corticotropin-releasing hormone-1 receptor. We demonstrated that an identified cholecystokinin (CCK)-positive NI neuronal population is part of the NI-MS tract, and that RLN3 and CCK NI neurons belong to a neuronal pool expressing the calcium-binding proteins, calbindin and calretinin. Finally, our electrophysiological studies revealed that MS is innervated by A-type potassium current-expressing, type I NI neurons, and that type I and II NI neurons differ markedly in their neurophysiological properties. Together these findings indicate that the MS is controlled by a discrete NI neuronal network with specific electrophysiological and neurochemical features; and these data are of particular importance for understanding neuronal mechanisms underlying the control of the septohippocampal system and related behaviors.

Relaxin-3 Innervation From the Nucleus Incertus to the Parahippocampal Cortex of the Rat.

Spatial learning and memory processes depend on anatomical and functional interactions between the hippocampus and the entorhinal cortex. A key neurophysiological component of these processes is hippocampal theta rhythm, which can be driven from subcortical areas including the pontine nucleus incertus (NI). The NI contains the largest population of neurons that produce and presumably release the neuropeptide, relaxin-3, which acts via the G i/o -protein-coupled receptor, relaxin-family peptide 3 receptor (RXFP3). NI activation induces general arousal including hippocampal theta, and inactivation induces impairment of spatial memory acquisition or retrieval. The primary aim of this study was to map the NI/relaxin-3 innervation of the parahippocampal cortex (PHC), including the medial and lateral entorhinal cortex, endopiriform cortex, perirhinal, postrhinal, and ectorhinal cortex, the amygdalohippocampal transition area and posteromedial cortical amygdala. Retrograde tracer injections were placed in different parts of the medial and lateral entorhinal cortex, which produced prominent retrograde labeling in the ipsilateral NI and some labeling in the contralateral NI. Anterograde tracer injections into the NI and immunostaining for relaxin-3 produced fiber labeling in deep layers of all parahippocampal areas and some dispersed fibers in superficial layers. Double-labeling studies revealed that both hippocampal projecting and calcium-binding protein-positive (presumed GABAergic) neurons received a relaxin-3 NI innervation. Some of these fibers also displayed synaptophysin (Syn) immunoreactivity, consistent with the presence of the peptide at synapses; and relaxin-3-positive fibers containing Syn bouton-like staining were frequently observed in contact with hippocampal-projecting or calcium-binding protein-positive neuronal somata and more distal elements. Finally, in situ hybridization studies revealed that entorhinal neurons in the superficial layers, and to a lesser extent in deep layers, contain RXFP3 mRNA. Together, our data support functional actions of the NI/relaxin-3-parahippocampal innervation on processes related to memory, spatial navigation and contextual analysis.

Analgesic effect of central relaxin receptor activation on persistent inflammatory pain in mice: behavioral and neurochemical data.

INTRODUCTION: The relaxin peptide signaling system is involved in diverse physiological processes, but its possible roles in the brain, including nociception, are largely unexplored. OBJECTIVE: In light of abundant expression of relaxin receptor (RXFP1) mRNA/protein in brain regions involved in pain processing, we investigated the effects of central RXFP1 activation on nociceptive behavior in a mouse model of inflammatory pain and examined the neurochemical phenotype and connectivity of relaxin and RXFP1 mRNA-positive neurons. METHODS: Mice were injected with Complete Freund Adjuvant (CFA) into a hind paw. After 4 days, the RXFP1 agonist peptides, H2-relaxin or B7-33, ± the RXFP1 antagonist, B-R13/17K-H2, were injected into the lateral cerebral ventricle, and mechanical and thermal sensitivity were assessed at 30 to 120 minutes. Relaxin and RXFP1 mRNA in excitatory and inhibitory neurons were examined using multiplex, fluorescent in situ hybridization. Relaxin-containing neurons were detected using immunohistochemistry and their projections assessed using fluorogold retrograde tract-tracing. RESULTS: Both H2-relaxin and B7-33 produced a strong, but transient, reduction in mechanical and thermal sensitivity of the CFA-injected hind paw alone, at 30 minutes postinjection. Notably, coinjection of B-R13/17K-H2 blocked mechanical, but not thermal, analgesia. In the claustrum, cingulate cortex, and subiculum, RXFP1 mRNA was expressed in excitatory neurons. Relaxin immunoreactivity was detected in neurons in forebrain and midbrain areas involved in pain processing and sending projections to the RXFP1-rich, claustrum and cingulate cortex. No changes were detected in CFA mice. CONCLUSION: Our study identified a previously unexplored peptidergic system that can control pain processing in the brain and produce analgesia.

Molecular Mechanisms Underlying the Beneficial Effects of Exercise on Brain Function and Neurological Disorders.

As life expectancy has increased, particularly in developed countries, due to medical advances and increased prosperity, age-related neurological diseases and mental health disorders have become more prevalent health issues, reducing the well-being and quality of life of sufferers and their families. In recent decades, due to reduced work-related levels of physical activity, and key research insights, prescribing adequate exercise has become an innovative strategy to prevent or delay the onset of these pathologies and has been demonstrated to have therapeutic benefits when used as a sole or combination treatment. Recent evidence suggests that the beneficial effects of exercise on the brain are related to several underlying mechanisms related to muscle-brain, liver-brain and gut-brain crosstalk. Therefore, this review aims to summarize the most relevant current knowledge of the impact of exercise on mood disorders and neurodegenerative diseases, and to highlight the established and potential underlying mechanisms involved in exercise-brain communication and their benefits for physiology and brain function.

Analysis of morphological and neurochemical changes in subthalamic nucleus neurons in response to a unilateral 6-OHDA lesion of the substantia nigra in adult rats.

BACKGROUND: Subthalamic nucleus (STN) neurons undergo changes in their pattern of activity and morphology during the clinical course of Parkinson's disease (PD). Striatal dopamine depletion and hyperactivity of neurons in the parafascicular nucleus (Pf) of the intralaminar thalamus are predicted to contribute to the STN changes. OBJECTIVE: This study investigated possible morphological and neurochemical changes in STN neurons in a rat model of unilateral, nigral dopamine neuron loss, in relation to previously documented alterations in Pf neurons. METHODS: Male Sprague-Dawley rats received a unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta (SNpc). Rats were randomly divided into two groups (6/group) for study at 1 and 5 months by post-treatment. The extent of SNpc dopamine neuron damage was assessed in an amphetamine-induced rotation test and postmortem assessment of tyrosine hydroxylase mRNA levels using in situ hybridization histochemistry. Neural cross-sectional measurements and assessment of vesicular glutamate transporter-2 (vGlut2) mRNA levels were performed to measure the impact on neurons in the STN. RESULTS: A unilateral SNpc dopaminergic neuron lesion significantly decreased the cross-sectional area of STN neurons ipsilateral to the lesion, at 1 month (P < 0.05) and 5 months (P < 0.01) post-lesion, while bilateral vGlut2 mRNA levels in STN neurons were unaltered. CONCLUSIONS: Decreased size of STN neurons in the presence of sustained vGlut2 mRNA levels following a unilateral SNpc 6-OHDA lesion, indicate altered STN physiology. This study presents further details of changes within the STN, coincident with observed alterations in Pf neurons and behaviour. DATA AVAILABILITY: The data associated with the findings of this study are available from the corresponding author upon request.

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.

Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action.

INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

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.

Regulatory peptides and systems biology: A new era of translational and reverse-translational neuroendocrinology.

Recently, there has been a resurgence in regulatory peptide science as a result of three converging trends. The first is the increasing population of the drug pipeline with peptide-based therapeutics, mainly in, but not restricted to, incretin-like molecules for treatment of metabolic disorders such as diabetes. The second is the development of genetic and optogenetic tools enabling new insights into how peptides actually function within brain and peripheral circuits to accomplish homeostatic and allostatic regulation. The third is the explosion in defined structures of the G-protein coupled receptors to which most regulatory peptides bind and exert their actions. These trends have closely wedded basic systems biology to drug discovery and development, creating a "two-way street" on which translational advances travel from basic research to the clinic, and, equally importantly, "reverse-translational" information is gathered, about the molecular, cellular and circuit-level mechanisms of action of regulatory peptides, comprising information required for the fine-tuning of drug development through testing in animal models. This review focuses on a small group of 'influential' peptides, including oxytocin, vasopressin, pituitary adenylate cyclase-activating polypeptide, ghrelin, relaxin-3 and glucagon-like peptide-1, and how basic discoveries and their application to therapeutics have intertwined over the past decade.