Publications by authors named "Sherie Ma"

40 Publications

In the Loop: Extrastriatal Regulation of Spiny Projection Neurons by GPR52.

ACS Chem Neurosci 2020 07 7;11(14):2066-2076. Epub 2020 Jul 7.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010, Australia.

GPR52 is a Gα-coupled orphan receptor identified as a putative target for the treatment of schizophrenia. The unique expression and signaling profile of GPR52 in key areas of dopamine and glutamate dysregulation suggests its activation may resolve both cortical and striatal dysfunction in the disorder. GPR52 mRNA is enriched in the striatum, almost exclusively on dopamine D-expressing medium spiny neurons (MSNs), and to a lesser extent in the cortex, predominantly on D-expressing pyramidal neurons. Synthetic, small molecule GPR52 agonists are effective in preclinical models of psychosis; however, the relative contribution of cortical and striatal GPR52 is unknown. Here we show that the GPR52 agonist, 3-BTBZ, inhibits phencyclidine-induced hyperlocomotor activity to a greater degree than amphetamine-induced motor effects, suggesting a mechanism beyond functional antagonism of striatal dopamine D receptor signaling. Using DARPP-32 phosphorylation and electrophysiological recordings in either striatopallidal or striatonigral MSNs, we were surprised to find no significant effect of 3-BTBZ in striatopallidal MSNs, but GPR52-mediated effects in striatonigral MSNs, where its mRNA is absent. 3-BTBZ increases phosphorylation of T75 on DARPP-32 in striatonigral MSNs, an effect that was dependent on cortical inputs. A similar role for GPR52 in regulating extrastriatal glutamatergic drive onto striatonigral MSNs was also evident in recordings of spontaneous excitatory postsynaptic currents and was shown to be dependent on the metabotropic glutamate (mGlu) receptor subtype 1. Our results demonstrate that GPR52-mediated regulation of striatal function depends heavily on extrastriatal inputs, which may further support its utility as a novel target for the treatment of schizophrenia.
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http://dx.doi.org/10.1021/acschemneuro.0c00197DOI Listing
July 2020

Effects of chronic silencing of relaxin-3 production in nucleus incertus neurons on food intake, body weight, anxiety-like behaviour and limbic brain activity in female rats.

Psychopharmacology (Berl) 2020 Apr 3;237(4):1091-1106. Epub 2020 Jan 3.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia.

Eating disorders are frequently triggered by stress and are more prevalent in women than men. First signs often appear during early adolescence, but the biological basis for the sex-specific differences is unknown. Central administration of native relaxin-3 (RLN3) peptide or chimeric/truncated analogues produces differential effects on food intake and HPA axis activity in adult male and female rats, but the precise role of endogenous RLN3 signalling in metabolic and neuroendocrine control is unclear. Therefore, we examined the effects of microRNA-induced depletion (knock-down) of RLN3 mRNA/(peptide) production in neurons of the brainstem nucleus incertus (NI) in female rats on a range of physiological, behavioural and neurochemical indices, including food intake, body weight, anxiety, plasma corticosterone, mRNA levels of key neuropeptides in the paraventricular nucleus of hypothalamus (PVN) and limbic neural activity patterns (reflected by c-fos mRNA). Validated depletion of RLN3 in NI neurons of female rats (n = 8) produced a small, sustained (~ 2%) decrease in body weight, an imbalance in food intake and an increase in anxiety-like behaviour in the large open field, but not in the elevated plus-maze or light/dark box. Furthermore, NI RLN3 depletion disrupted corticosterone regulation, increased oxytocin and arginine-vasopressin, but not corticotropin-releasing factor, mRNA, in PVN, and decreased basal levels of c-fos mRNA in parvocellular and magnocellular PVN, bed nucleus of stria terminalis and the lateral hypothalamic area, brain regions involved in stress and feeding. These findings support a role for NI RLN3 neurons in fine-tuning stress and neuroendocrine responses and food intake regulation in female rats.
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http://dx.doi.org/10.1007/s00213-019-05439-1DOI Listing
April 2020

Targeted viral vector transduction of relaxin-3 neurons in the rat using a novel cell-type specific promoter.

IBRO Rep 2020 Jun 13;8:1-10. Epub 2019 Dec 13.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia.

Modern neuroscience utilizes transgenic techniques extensively to study the activity and function of brain neural networks. A key feature of this approach is its compatibility with molecular methods for selective transgene expression in neuronal circuits of interest. Until now, such targeted transgenic approaches have not been applied to the extensive circuitry involving the neuropeptide, relaxin-3. Pharmacological and gene knock-out studies have revealed relaxin-3 signalling modulates interrelated behaviours and cognitive processes, including stress and anxiety, food and alcohol consumption, and spatial and social memory, highlighting the potential of this system as a therapeutic target. In the present study, we aimed to identify a promoter sequence capable of regulating cell-type specific transgene expression from an adeno-associated viral (AAV) vector in relaxin-3 neurons of the rat (NI). In parallel to relaxin-3 promoter sequences, we also tested an AAV vector containing promoter elements for the tropomyosin receptor kinase A (TrkA) gene, as TrkA is co-expressed with relaxin-3 in rat NI neurons. Stereotaxic injection of an mCherry-expressing AAV vector revealed widespread non-specific TrkA promoter (880 bp) activity in and adjacent to the NI at 8 weeks post-treatment. In contrast, mCherry expression was successfully restricted to relaxin-3 NI neurons with 98% specificity using a 1736 bp relaxin-3 promoter. In addition to detailed anatomical mapping of NI relaxin-3 networks, illustrated here in association with GABAergic medial septum neurons, this method for targeted transgene delivery offers a versatile tool for ongoing preclinical studies of relaxin-3 circuitry.
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http://dx.doi.org/10.1016/j.ibror.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928288PMC
June 2020

Validation of 'Somnivore', a Machine Learning Algorithm for Automated Scoring and Analysis of Polysomnography Data.

Front Neurosci 2019 18;13:207. Epub 2019 Mar 18.

The Florey Institute of Neuroscience and Mental Health, Parkville, VIC, Australia.

Manual scoring of polysomnography data is labor-intensive and time-consuming, and most existing software does not account for subjective differences and user variability. Therefore, we evaluated a supervised machine learning algorithm, Somnivore, for automated wake-sleep stage classification. We designed an algorithm that extracts features from various input channels, following a brief session of manual scoring, and provides automated wake-sleep stage classification for each recording. For algorithm validation, polysomnography data was obtained from independent laboratories, and include normal, cognitively-impaired, and alcohol-treated human subjects (total = 52), narcoleptic mice and drug-treated rats (total = 56), and pigeons ( = 5). Training and testing sets for validation were previously scored manually by 1-2 trained sleep technologists from each laboratory. -measure was used to assess precision and sensitivity for statistical analysis of classifier output and human scorer agreement. The algorithm gave high concordance with manual visual scoring across all human data (wake 0.91 ± 0.01; N1 0.57 ± 0.01; N2 0.81 ± 0.01; N3 0.86 ± 0.01; REM 0.87 ± 0.01), which was comparable to manual inter-scorer agreement on all stages. Similarly, high concordance was observed across all rodent (wake 0.95 ± 0.01; NREM 0.94 ± 0.01; REM 0.91 ± 0.01) and pigeon (wake 0.96 ± 0.006; NREM 0.97 ± 0.01; REM 0.86 ± 0.02) data. Effects of classifier learning from single signal inputs, simple stage reclassification, automated removal of transition epochs, and training set size were also examined. In summary, we have developed a polysomnography analysis program for automated sleep-stage classification of data from diverse species. Somnivore enables flexible, accurate, and high-throughput analysis of experimental and clinical sleep studies.
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http://dx.doi.org/10.3389/fnins.2019.00207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431640PMC
March 2019

Chronic activation of the relaxin-3 receptor on GABA neurons in rat ventral hippocampus promotes anxiety and social avoidance.

Hippocampus 2019 10 19;29(10):905-920. Epub 2019 Mar 19.

Discovery Science, The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.

Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a G -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.
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http://dx.doi.org/10.1002/hipo.23089DOI Listing
October 2019

Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons.

Brain Struct Funct 2019 Jan 28;224(1):453-469. Epub 2018 Oct 28.

Departamento de Medicina, Facultad de Ciencias de la Salud, Universitat Jaume I, Av de Vicent Sos Baynat, s/n, 12071, Castellón de la Plana, Castellón, Spain.

In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the G-protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling.
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http://dx.doi.org/10.1007/s00429-018-1763-5DOI Listing
January 2019

Acquisition of analgesic properties by the cholecystokinin (CCK)/CCK2 receptor system within the amygdala in a persistent inflammatory pain condition.

Pain 2019 Feb;160(2):345-357

Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.

Pain is associated with negative emotions such as anxiety, but the underlying neurocircuitry and modulators of the association of pain and anxiety remain unclear. The neuropeptide cholecystokinin (CCK) has both pronociceptive and anxiogenic properties, so we explored the role of CCK in anxiety and nociception in the central amygdala (CeA), a key area in control of emotions and descending pain pathways. Local infusion of CCK into the CeA of control rats increased anxiety, as measured in the light-dark box test, but had no effect on mechanical sensitivity. By contrast, intra-CeA CCK infusion 4 days after Complete Freund's Adjuvant (CFA) injection into the hindpaw resulted in analgesia, but also in loss of its anxiogenic capacity. Inflammatory conditions induced changes in the CeA CCK signaling system with an increase of CCK immunoreactivity and a decrease in CCK1, but not CCK2, receptor mRNA. In CFA rats, patch-clamp experiments revealed that CCK infusion increased CeA neuron excitability. It also partially blocked the discharge of wide dynamic range neurons in the dorsal spinal cord. These effects of CCK on CeA and spinal neurons in CFA rats were mimicked by the specific CCK2 receptor agonist, gastrin. This analgesic effect was likely mediated by identified CeA neurons projecting to the periaqueductal gray matter that express CCK receptors. Together, our data demonstrate that intra-CeA CCK infusion activated a descending CCK2 receptor-dependent pathway that inhibited spinal neuron discharge. Thus, persistent pain induces a functional switch to a newly identified analgesic capacity of CCK in the amygdala, indicating central emotion-related circuit controls pain transmission in spinal cord.
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http://dx.doi.org/10.1097/j.pain.0000000000001408DOI Listing
February 2019

Involvement of Serotonergic and Relaxin-3 Neuropeptide Systems in the Expression of Anxiety-like Behavior.

Neuroscience 2018 10 18;390:88-103. Epub 2018 Aug 18.

School of Psychology and Public Health, La Trobe University, Bundoora, Victoria, Australia. Electronic address:

Anxiety-related defensive behavior is controlled by a distributed network of brain regions and interconnected neural circuits. The dorsal raphe nucleus (DR), which contains the majority of forebrain-projecting serotonergic neurons, is a key brain region involved in fear states and anxiety-related behavior via modulation of this broad neural network. Evidence suggests that relaxin-3 neurons in the nucleus incertus (NI) may also interact with this network, however, the potential role of the NI in the control of anxiety-related defensive behavior requires further investigation. In this study, we examined the response of an anxiety-related neuronal network, including serotonergic neurons in the DR and relaxin-3-containing neurons in the NI, to administration of an anxiogenic drug and exposure to an aversive environment. We administered an anxiogenic dose of the adenosine receptor antagonist, caffeine (50 mg/kg, i.p.), or vehicle, to adult male Wistar rats and 30 min later exposed them to either an elevated plus-maze (EPM) or a home cage environment. Administration of caffeine and exposure to the EPM activated a broad network of brain regions involved in control of anxiety-like behaviors, including serotonergic neurons in the DR, as measured using c-Fos immunohistochemistry. However, only exposure to the EPM activated relaxin-3-containing neurons in the NI, and activation of these neurons was not correlated with changes in anxiety-like behavior. These data suggest activation of the NI relaxin-3 system is associated with expression of behavior in tests of anxiety, but may not be directly involved in the approach-avoidance conflict inherent in anxiety-related defensive behavior in rodents.
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http://dx.doi.org/10.1016/j.neuroscience.2018.08.007DOI Listing
October 2018

Gram scale preparation of clozapine -oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs.

MethodsX 2018 23;5:257-267. Epub 2018 Mar 23.

School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia.

Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine -oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: •A simple extraction method of clozapine from commercial tablets;•A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and•That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq . This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.
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http://dx.doi.org/10.1016/j.mex.2018.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053635PMC
March 2018

Melanin-concentrating hormone and orexin systems in rat nucleus incertus: Dual innervation, bidirectional effects on neuron activity, and differential influences on arousal and feeding.

Neuropharmacology 2018 09 6;139:238-256. Epub 2018 Jul 6.

Department of Neurophysiology and Chronobiology, Jagiellonian University, Krakow, Poland. Electronic address:

The rat nucleus incertus (NI) contains GABA/peptide-projection neurons responsive to orexin (hypocretin)/orexin receptor-2 (OX) signalling. Melanin-concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor-1 (MCH) expression, and neurophysiological and behavioural effects of MCH c.f. orexin-A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub-zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH-containing fibres in NI. Single-cell RT-PCR detected MCH and OX mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co-expression patterns of MCH and OX mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch-clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1 μM), representing a distinct population from OXA-sensitive NI neurons (35%). Intra-NI OXA infusion (600 pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra-NI MCH infusion (600 pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre-fasted rats tested during the dark/active phase, intra-NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide-immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours.
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http://dx.doi.org/10.1016/j.neuropharm.2018.07.004DOI Listing
September 2018

Modulation of forebrain function by nucleus incertus and relaxin-3/RXFP3 signaling.

CNS Neurosci Ther 2018 08 2;24(8):694-702. Epub 2018 May 2.

The Florey Institute of Neuroscience and Mental Health, Parkville, Vic., Australia.

The nucleus incertus (NI) in the pontine tegmentum sends ascending projections to the midbrain, hypothalamus, amygdala, basal forebrain, hippocampus, and prefrontal cortex, and has a postulated role in modulating several forebrain functions. A substantial population of GABAergic NI neurons expresses the neuropeptide, relaxin-3, which acts via the G -protein-coupled receptor, RXFP3, present throughout the forebrain target regions. Broad and specific manipulations of these systems by activation or inhibition of the NI or modulating RXFP3 signaling have revealed key insights into the likely influence of the NI/relaxin-3/RXFP3 system on modalities including arousal, feeding, stress responses, anxiety and addiction, and attention and memory. This range of actions corresponds to a likely impact of NI/(relaxin-3) projections on multiple integrated circuits, but makes it difficult to draw conclusions about a generalized function for this network. This review will focus on the key physiological process of oscillatory theta rhythm and the neural circuits that promote it during behavioral activation, highlighting the ability of NI and relaxin-3/RXFP3 signaling systems to modulate these circuits. A better understanding of these mechanisms may provide a way to therapeutically adjust malfunction of forebrain activity present in several pathological conditions.
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http://dx.doi.org/10.1111/cns.12862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6490102PMC
August 2018

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA.

Front Neuroanat 2017 17;11:133. Epub 2018 Jan 17.

Unitat Predepartamental de Medicina, Facultat de Ciències de la Salut, Universitat Jaume I, Castellón, Spain.

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The G-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex hybridization to characterize the septal neurons expressing mRNA in the rat. Our results demonstrate that mRNA is abundantly expressed in vesicular GABA transporter () mRNA- and parvalbumin () mRNA-positive GABA neurons in MS, whereas mRNA-positive acetylcholine neurons lack mRNA. Approximately 75% of mRNA-positive neurons expressed mRNA (and 22% were mRNA-positive), while the remaining 25% expressed mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.
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http://dx.doi.org/10.3389/fnana.2017.00133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777284PMC
January 2018

Dual-transmitter systems regulating arousal, attention, learning and memory.

Neurosci Biobehav Rev 2018 02 27;85:21-33. Epub 2017 Jul 27.

The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia; Florey Department of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia; Department of Anatomy and Neuroscience, The University of Melbourne, Victoria, Australia. Electronic address:

An array of neuromodulators, including monoamines and neuropeptides, regulate most behavioural and physiological traits. In the past decade, dramatic progress has been made in mapping neuromodulatory circuits, in analysing circuit dynamics, and interrogating circuit function using pharmacogenetic, optogenetic and imaging methods This review will focus on several distinct neural networks (acetylcholine/GABA/glutamate; histamine/GABA; orexin/glutamate; and relaxin-3/GABA) that originate from neural hubs that regulate wakefulness and related attentional and cognitive processes, and highlight approaches that have identified dual transmitter roles in these behavioural functions. Modulation of these different neural networks might be effective treatments of diseases related to arousal/sleep dysfunction and of cognitive dysfunction in psychiatric and neurodegenerative disorders.
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http://dx.doi.org/10.1016/j.neubiorev.2017.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747977PMC
February 2018

Distribution, physiology and pharmacology of relaxin-3/RXFP3 systems in brain.

Br J Pharmacol 2017 05 4;174(10):1034-1048. Epub 2016 Dec 4.

The Florey Institute of Neuroscience and Mental Health, Parkville, Victoria, Australia.

Relaxin-3 is a member of a superfamily of structurally-related peptides that includes relaxin and insulin-like peptide hormones. Soon after the discovery of the relaxin-3 gene, relaxin-3 was identified as an abundant neuropeptide in brain with a distinctive topographical distribution within a small number of GABAergic neuron populations that is well conserved across species. Relaxin-3 is thought to exert its biological actions through a single class-A GPCR - relaxin-family peptide receptor 3 (RXFP3). Class-A comprises GPCRs for relaxin-3 and insulin-like peptide-5 and other peptides such as orexin and the monoamine transmitters. The RXFP3 receptor is selectively activated by relaxin-3, whereas insulin-like peptide-5 is the cognate ligand for the related RXFP4 receptor. Anatomical and pharmacological evidence obtained over the last decade supports a function of relaxin-3/RXFP3 systems in modulating responses to stress, anxiety-related and motivated behaviours, circadian rhythms, and learning and memory. Electrophysiological studies have identified the ability of RXFP3 agonists to directly hyperpolarise thalamic neurons in vitro, but there are no reports of direct cell signalling effects in vivo. This article provides an overview of earlier studies and highlights more recent research that implicates relaxin-3/RXFP3 neural network signalling in the integration of arousal, motivation, emotion and related cognition, and that has begun to identify the associated neural substrates and mechanisms. Future research directions to better elucidate the connectivity and function of different relaxin-3 neuron populations and their RXFP3-positive target neurons in major experimental species and humans are also identified.

Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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http://dx.doi.org/10.1111/bph.13659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406293PMC
May 2017

Relaxin' the brain: a case for targeting the nucleus incertus network and relaxin-3/RXFP3 system in neuropsychiatric disorders.

Br J Pharmacol 2017 05 6;174(10):1061-1076. Epub 2016 Sep 6.

Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

Relaxin-3 has been proposed to modulate emotional-behavioural functions such as arousal and behavioural activation, appetite regulation, stress responses, anxiety, memory, sleep and circadian rhythm. The nucleus incertus (NI), in the midline tegmentum close to the fourth ventricle, projects widely throughout the brain and is the primary site of relaxin-3 neurons. Over recent years, a number of preclinical studies have explored the function of the NI and relaxin-3 signalling, including reports of mRNA or peptide expression changes in the NI in response to behavioural or pharmacological manipulations, effects of lesions or electrical or pharmacological manipulations of the NI, effects of central microinfusions of relaxin-3 or related agonist or antagonist ligands on physiology and behaviour, and the impact of relaxin-3 gene deletion or knockdown. Although these individual studies reveal facets of the likely functional relevance of the NI and relaxin-3 systems for human physiology and behaviour, the differences observed in responses between species (e.g. rat vs. mouse), the clearly identified heterogeneity of NI neurons and procedural differences between laboratories are some of the factors that have prevented a precise understanding of their function. This review aims to draw attention to the current preclinical evidence available that suggests the relevance of the NI/relaxin-3 system to the pathology and/or symptoms of certain neuropsychiatric disorders and to provide cognizant directions for future research to effectively and efficiently uncover its therapeutic potential.

Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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http://dx.doi.org/10.1111/bph.13564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406295PMC
May 2017

Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

J Med Chem 2016 08 8;59(16):7445-56. Epub 2016 Aug 8.

Florey Institute of Neuroscience and Mental Health and Florey Department of Neuroscience and Mental Health, University of Melbourne , Melbourne, Victoria 3052, Australia.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00265DOI Listing
August 2016

Nucleus incertus promotes cortical desynchronization and behavioral arousal.

Brain Struct Funct 2017 01 20;222(1):515-537. Epub 2016 May 20.

The Florey Institute of Neuroscience and Mental Health, 30 Royal Parade, Parkville, VIC, 3052, Australia.

Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach-clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs-to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.
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http://dx.doi.org/10.1007/s00429-016-1230-0DOI Listing
January 2017

Comparative Distribution of Relaxin-3 Inputs and Calcium-Binding Protein-Positive Neurons in Rat Amygdala.

Front Neuroanat 2016 7;10:36. Epub 2016 Apr 7.

Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Universitat ValenciaValencia, Spain; Unitat Predepartamental de Medicina, Universitat Jaume ICastellón, Spain.

The neural circuits involved in mediating complex behaviors are being rapidly elucidated using various newly developed and powerful anatomical and molecular techniques, providing insights into the neural basis for anxiety disorders, depression, addiction, and dysfunctional social behaviors. Many of these behaviors and associated physiological processes involve the activation of the amygdala in conjunction with cortical and hippocampal circuits. Ascending subcortical projections provide modulatory inputs to the extended amygdala and its related nodes (or "hubs") within these key circuits. One such input arises from the nucleus incertus (NI) in the tegmentum, which sends amino acid- and peptide-containing projections throughout the forebrain. Notably, a distinct population of GABAergic NI neurons expresses the highly-conserved neuropeptide, relaxin-3, and relaxin-3 signaling has been implicated in the modulation of reward/motivation and anxiety- and depressive-like behaviors in rodents via actions within the extended amygdala. Thus, a detailed description of the relaxin-3 innervation of the extended amygdala would provide an anatomical framework for an improved understanding of NI and relaxin-3 modulation of these and other specific amygdala-related functions. Therefore, in this study, we examined the distribution of NI projections and relaxin-3-positive elements (axons/fibers/terminals) within the amygdala, relative to the distribution of neurons expressing the calcium-binding proteins, parvalbumin (PV), calretinin (CR) and/or calbindin. Anterograde tracer injections into the NI revealed a topographic distribution of NI efferents within the amygdala that was near identical to the distribution of relaxin-3-immunoreactive fibers. Highest densities of anterogradely-labeled elements and relaxin-3-immunoreactive fibers were observed in the medial nucleus of the amygdala, medial divisions of the bed nucleus of the stria terminalis (BST) and in the endopiriform nucleus. In contrast, sparse anterogradely-labeled and relaxin-3-immunoreactive fibers were observed in other amygdala nuclei, including the lateral, central and basal nuclei, while the nucleus accumbens lacked any innervation. Using synaptophysin as a synaptic marker, we identified relaxin-3 positive synaptic terminals in the medial amygdala, BST and endopiriform nucleus of amygdala. Our findings demonstrate the existence of topographic NI and relaxin-3-containing projections to specific nuclei of the extended amygdala, consistent with a likely role for this putative integrative arousal system in the regulation of amygdala-dependent social and emotional behaviors.
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http://dx.doi.org/10.3389/fnana.2016.00036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4823275PMC
April 2016

Septal projections to nucleus incertus in the rat: bidirectional pathways for modulation of hippocampal function.

J Comp Neurol 2015 Mar 19;523(4):565-88. Epub 2014 Dec 19.

Departamento de Medicina, Universitat Jaume I, 12071, Castellón, Spain.

Projections from the nucleus incertus (NI) to the septum have been implicated in the modulation of hippocampal theta rhythm. In this study we describe a previously uncharacterized projection from the septum to the NI, which may provide feedback modulation of the ascending circuitry. Fluorogold injections into the NI resulted in retrograde labeling in the septum that was concentrated in the horizontal diagonal band and areas of the posterior septum including the septofimbrial and triangular septal nuclei. Double-immunofluorescent staining indicated that the majority of NI-projecting septal neurons were calretinin-positive and some were parvalbumin-, calbindin-, or glutamic acid decarboxylase (GAD)-67-positive. Choline acetyltransferase-positive neurons were Fluorogold-negative. Injection of anterograde tracers into medial septum, or triangular septal and septofimbrial nuclei, revealed fibers descending to the supramammillary nucleus, median raphe, and the NI. These anterogradely labeled varicosities displayed synaptophysin immunoreactivity, indicating septal inputs form synapses on NI neurons. Anterograde tracer also colocalized with GAD-67-positive puncta in labeled fibers, which in some cases made close synaptic contact with GAD-67-labeled NI neurons. These data provide evidence for the existence of an inhibitory descending projection from medial and posterior septum to the NI that provides a "feedback loop" to modulate the comparatively more dense ascending NI projections to medial septum and hippocampus. Neural processes and associated behaviors activated or modulated by changes in hippocampal theta rhythm may depend on reciprocal connections between ascending and descending pathways rather than on unidirectional regulation via the medial septum.
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http://dx.doi.org/10.1002/cne.23687DOI Listing
March 2015

Relaxin-3/RXFP3 Signaling and Neuroendocrine Function - A Perspective on Extrinsic Hypothalamic Control.

Front Endocrinol (Lausanne) 2013 Sep 18;4:128. Epub 2013 Sep 18.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne , Melbourne, VIC , Australia ; Department of Biochemistry and Molecular Biology, The University of Melbourne , Melbourne, VIC , Australia.

Complex neural circuits within the hypothalamus that govern essential autonomic processes and associated behaviors signal using amino acid and monoamine transmitters and a variety of neuropeptide (hormone) modulators, often via G-protein coupled receptors (GPCRs) and associated cellular pathways. Relaxin-3 is a recently identified neuropeptide that is highly conserved throughout evolution. Neurons expressing relaxin-3 are located in the brainstem, but broadly innervate the entire limbic system including the hypothalamus. Extensive anatomical data in rodents and non-human primate, and recent regulatory and functional data, suggest relaxin-3 signaling via its cognate GPCR, RXFP3, has a broad range of effects on neuroendocrine function associated with stress responses, feeding and metabolism, motivation and reward, and possibly sexual behavior and reproduction. Therefore, this article aims to highlight the growing appreciation of the relaxin-3/RXFP3 system as an important "extrinsic" regulator of the neuroendocrine axis by reviewing its neuroanatomy and its putative roles in arousal-, stress-, and feeding-related behaviors and links to associated neural substrates and signaling networks. Current evidence identifies RXFP3 as a potential therapeutic target for treatment of neuroendocrine disorders and related behavioral dysfunction.
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http://dx.doi.org/10.3389/fendo.2013.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776160PMC
September 2013

Heterogeneous responses of nucleus incertus neurons to corticotrophin-releasing factor and coherent activity with hippocampal theta rhythm in the rat.

J Physiol 2013 Aug 13;591(16):3981-4001. Epub 2013 May 13.

The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria 3010, Australia.

The nucleus incertus (NI) of the rat hindbrain is a putative node in the ascending control of the septohippocampal system and hippocampal theta rhythm and is stress and arousal responsive. NI contains GABA neurons that express multiple neuropeptides, including relaxin-3 (RLN3) and neuropeptide receptors, including corticotrophin-releasing factor receptor-1 (CRF-R1), but the precise anatomical and physiological characteristics of NI neurons are unclear. Therefore, we examined the firing properties of NI neurons and their responses to CRF, the correlation of these responses with occurrence of relaxin-3, and NI neuron morphology in the rat. Most NI neurons excited by intracerebroventricular CRF infusion were RLN3-positive (9 of 10), whereas all inhibited cells were RLN3-negative (8 of 8). The spontaneous firing of RLN3 (n = 6) but not non-RLN3 neurons (n = 6) was strongly modulated and phase-locked with the initial ascending phase of hippocampal theta oscillations. In brain slices, the majority of recorded NI neurons (15 of 19) displayed excitatory responses to CRF, which uniformly increased action potential frequency and membrane potential depolarization in the presence of tetrodotoxin, indicating a direct, postsynaptic action of CRF on NI neurons. This excitation was associated with reduction in the slow component of afterhyperpolarization and a strong depolarization. Quantitative analysis in naïve rats of validated CRF-R1, RLN3 and neuronal nuclear antigen (NeuN) immunoreactivity revealed 52% of NI neurons as CRF-R1 positive, of which 53% were RLN3 positive, while 48% of NI neurons lacked CRF-R1 and RLN3. All RLN3 neurons expressed CRF-R1. CRF neurons that projected to the NI were identified in lateral preoptic hypothalamus, but not in paraventricular hypothalamus, bed nucleus of stria terminalis or central amygdala. Our findings suggest NI is an important site for CRF modulation of hippocampal theta rhythm via effects on GABA/RLN3 transmission.
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http://dx.doi.org/10.1113/jphysiol.2013.254300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764641PMC
August 2013

Silencing relaxin-3 in nucleus incertus of adult rodents: a viral vector-based approach to investigate neuropeptide function.

PLoS One 2012 2;7(8):e42300. Epub 2012 Aug 2.

Department of Biochemistry and Molecular Biology, The University of Melbourne, Victoria, Australia.

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042300PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410922PMC
January 2013

Distribution and targets of the relaxin-3 innervation of the septal area in the rat.

J Comp Neurol 2012 Jun;520(9):1903-39

Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Universidad de Valencia, 46010 Valencia, Spain.

Neural tracing studies have revealed that the rat medial and lateral septum are targeted by ascending projections from the nucleus incertus, a population of tegmental GABA neurons. These neurons express the relaxin-family peptide, relaxin-3, and pharmacological modulation of relaxin-3 receptors in medial septum alters hippocampal theta rhythm and spatial memory. In an effort to better understand the basis of these interactions, we have characterized the distribution of relaxin-3 fibers/terminals in relation to different septal neuron populations identified using established protein markers. Dense relaxin-3 fiber plexuses were observed in regions of medial septum containing hippocampal-projecting choline acetyltransferase (ChAT)-, neuronal nitric oxide synthase (nNOS)-, and parvalbumin (PV)-positive neurons. In lateral septum (LS), relaxin-3 fibers were concentrated in the ventrolateral nucleus of rostral LS and the ventral nucleus of caudal LS, with sparse labeling in the dorsolateral and medial nuclei of rostral LS, dorsal nucleus of caudal LS, and ventral portion nuclei. Relaxin-3 fibers were also observed in the septofimbrial and triangular septal nuclei. In the medial septum, we observed relaxin-3-immunoreactive contacts with ChAT-, PV-, and glutamate decarboxylase-67-positive neurons that projected to hippocampus, and contacts between relaxin-3 terminals and calbindin- and calretinin-positive neurons. Relaxin-3 colocalized with synaptophysin in nerve terminals in all septal areas, and ultrastructural analysis revealed these terminals were symmetrical and contacted spines, somata, dendritic shafts, and occasionally other axonal terminals. These data predict that this GABA/peptidergic projection modulates septohippocampal activity and hippocampal theta rhythm related to exploratory navigation, defensive and ingestive behaviors, and responses to neurogenic stressors.
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http://dx.doi.org/10.1002/cne.23018DOI Listing
June 2012

Relaxin-3 systems in the brain--the first 10 years.

J Chem Neuroanat 2011 Dec 15;42(4):262-75. Epub 2011 Jun 15.

Florey Neuroscience Institutes, The University of Melbourne, Victoria, Australia.

The relaxin-3 gene was identified in 2001 by searching the human genome database for homologues of the relaxin hormone, and was subsequently discovered to encode a highly conserved neuropeptide in mammals and lower species. In the decade since its discovery there have been significant advances in our knowledge of the peptide, including the identification of its cognate receptor (a type 1 G-protein coupled receptor, GPCR135 or RXFP3), an understanding of its structure-activity and associated cellular signalling, and the elucidation of key neuroanatomical aspects of relaxin-3/RXFP3 networks in mammalian brain. The latter studies revealed that relaxin-3 is expressed within GABA neurons of the brainstem including an area known as the nucleus incertus, and that ascending relaxin-3 projections innervate a broad range of RXFP3-rich forebrain areas. These maps provided a foundation for pharmacological and physiological studies to elucidate the neurobiological nature of relaxin-3/RXFP3 signalling in vivo. Recent findings from our laboratory and others suggest the relaxin-3 neural network represents a newly identified ascending arousal system, able to modulate a range of interrelated functions including responses to stress, spatial and emotional memory, feeding and metabolism, motivation and reward, and circadian rhythm and sleep/wake states. More research is now required to discover further important facts about relaxin-3 neurons, such as their various regulatory inputs, and to characterise populations of RXFP3-positive neurons and determine their influence on particular neural circuits, physiology and complex behaviour.
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http://dx.doi.org/10.1016/j.jchemneu.2011.05.013DOI Listing
December 2011

Nucleus incertus--an emerging modulatory role in arousal, stress and memory.

Neurosci Biobehav Rev 2011 May 15;35(6):1326-41. Epub 2011 Feb 15.

Howard Florey Institute and Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia.

A major challenge in systems neuroscience is to determine the underlying neural circuitry and associated neurotransmitters and receptors involved in psychiatric disorders, such as anxiety and depression. A focus of many of these studies has been specific brainstem nuclei that modulate levels of arousal via their ascending monoaminergic projections (e.g. the serotonergic dorsal raphé, noradrenergic locus ceruleus and cholinergic laterodorsal tegmental nucleus). After years of relative neglect, the subject of recent studies in this context has been the GABAergic nucleus incertus, which is located in the midline periventricular central gray in the 'prepontine' hindbrain, with broad projections throughout the forebrain. Nucleus incertus neurons express receptors for the stress hormone, corticotropin-releasing factor (CRF), are activated by psychological stressors, and project to key nuclei involved in stress responses and behavioral activation. The nucleus incertus is also a node in neural circuits capable of modulating hippocampal theta rhythm, which is related to control of spatial navigation and memory. A significant population of nucleus incertus neurons express the recently discovered, highly conserved neuropeptide, relaxin-3; and the recent availability of structurally-related, chimeric peptides that selectively activate or inhibit the relaxin-3 receptor, RXFP3, is facilitating studies of relaxin-3/RXFP3 networks and associated GABA and CRF systems. It is predicted that such targeted research will help elucidate the functions of ascending nucleus incertus pathways, including their possible involvement in arousal (sleep/wakefulness), stress reponses, and learning and memory; and in the pathology of related psychiatric diseases such as insomnia, anxiety and depression, and cognitive deficits.
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http://dx.doi.org/10.1016/j.neubiorev.2011.02.004DOI Listing
May 2011

Distribution of relaxin-3 and RXFP3 within arousal, stress, affective, and cognitive circuits of mouse brain.

J Comp Neurol 2010 Oct;518(19):4016-45

Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia.

Relaxin-3 (RLN3) and its native receptor, relaxin family peptide 3 receptor (RXFP3), constitute a newly identified neuropeptide system enriched in mammalian brain. The distribution of RLN3/RXFP3 networks in rat brain and recent experimental studies suggest a role for this system in modulation of arousal, stress, metabolism, and cognition. In order to facilitate exploration of the biology of RLN3/RXFP3 in complementary murine models, this study mapped the neuroanatomical distribution of the RLN3/RXFP3 system in mouse brain. Adult, male wildtype and RLN3 knock-out (KO)/LacZ knock-in (KI) mice were used to map the central distribution of RLN3 gene expression and RLN3-like immunoreactivity (-LI). The distribution of RXFP3 mRNA and protein was determined using [(35)S]-oligonucleotide probes and a radiolabeled RXFP3-selective agonist ([(125)I]-R3/I5), respectively. High densities of neurons expressing RLN3 mRNA, RLN3-associated beta-galactosidase activity and RLN3-LI were detected in the nucleus incertus (or nucleus O), while smaller populations of positive neurons were observed in the pontine raphé, the periaqueductal gray and a region adjacent to the lateral substantia nigra. RLN3-LI was observed in nerve fibers/terminals in nucleus incertus and broadly throughout the pons, midbrain, hypothalamus, thalamus, septum, hippocampus, and neocortex, but was absent in RLN3 KO/LacZ KI mice. This RLN3 neural network overlapped the regional distribution of RXFP3 mRNA and [(125)I]-R3/I5 binding sites in wildtype and RLN3 KO/LacZ KI mice. These findings provide further evidence for the conserved nature of RLN3/RXFP3 systems in mammalian brain and the ability of RLN3/RXFP3 signaling to modulate "behavioral state" and an array of circuits involved in arousal, stress responses, affective state, and cognition.
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http://dx.doi.org/10.1002/cne.22442DOI Listing
October 2010

Modulation of hippocampal theta oscillations and spatial memory by relaxin-3 neurons of the nucleus incertus.

Learn Mem 2009 Nov 30;16(11):730-42. Epub 2009 Oct 30.

Howard Florey Institute, The University of Melbourne, Victoria 3010, Australia.

Hippocampal theta rhythm is thought to underlie learning and memory, and it is well established that "pacemaker" neurons in medial septum (MS) modulate theta activity. Recent studies in the rat demonstrated that brainstem-generated theta rhythm occurs through a multisynaptic pathway via the nucleus incertus (NI), which is the primary source of the neuropeptide relaxin-3 (RLN3). Therefore, this study examined the possible contribution of RLN3 to MS activity, and associated hippocampal theta activity and spatial memory. In anesthetized and conscious rats, we identified the ability of intraseptal RLN3 signaling to modulate neuronal activity in the MS and hippocampus and promote hippocampal theta rhythm. Behavioral studies in a spontaneous alternation task indicated that endogenous RLN3 signaling within MS promoted spatial memory and exploratory activity significantly increased c-Fos immunoreactivity in RLN3-producing NI neurons. Anatomical studies demonstrated axons/terminals from NI/RLN3 neurons make close contact with septal GABAergic (and cholinergic) neurons, including those that project to the hippocampus. In summary, RLN3 neurons of the NI can modulate spatial memory and underlying hippocampal theta activity through axonal projections to pacemaker neurons of the MS. NI/RLN3 neurons are highly responsive to stress and express corticotropin-releasing factor type-1 receptors, suggesting that the effects observed could be an important component of memory processing associated with stress responses.
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http://dx.doi.org/10.1101/lm.1438109DOI Listing
November 2009

Localization of relaxin-3 in brain of Macaca fascicularis: identification of a nucleus incertus in primate.

J Comp Neurol 2009 Dec;517(6):856-72

Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia.

Relaxin-3 (RLN3) is a highly conserved, ancestral member of the insulin/relaxin peptide family. RLN3 mRNA is highly expressed in rat, mouse, and human brain and molecular genetic and pharmacological studies suggest that RLN3 is the cognate ligand for the relaxin family peptide-3 receptor (RXFP3). The distribution of RLN3/RXFP3 networks has been determined in rat and mouse brain, but not in higher species. In this study we describe the distribution of RLN3 neurons in the brain of macaque (Macaca fascicularis) using in situ hybridization histochemistry and immunohistochemistry. RLN3 mRNA and high levels of RLN3-like immunoreactivity (-LI) were observed in neurons within a ventromedial region of the central gray of the pons and medulla that appears to represent the primate analog of the nucleus incertus (NI) described in lower species. Nerve fibers and terminals containing RLN3-LI were observed throughout brain regions identical to those known to receive afferents from the NI in the rat, including the septum, hippocampus, entorhinal cortex, lateral, dorsomedial and ventromedial hypothalamus, supramammillary and interpeduncular nuclei, anterodorsal, paraventricular and reuniens thalamic nuclei, lateral habenula, central gray, and dorsal raphe, solitary tract, and ambiguus nuclei. Experimental studies in the rat strongly implicate a role of this neuropeptide-receptor system in arousal, feeding, and metabolism, learning and memory, and central responses to psychological stressors. These new anatomical findings support the proposition that the RLN3 system is similarly involved in the integration and modulation of behavioral activation and arousal and responses to stress in nonhuman primates and humans.
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http://dx.doi.org/10.1002/cne.22197DOI Listing
December 2009

Swim stress excitation of nucleus incertus and rapid induction of relaxin-3 expression via CRF1 activation.

Neuropharmacology 2010 Jan 26;58(1):145-55. Epub 2009 Jun 26.

Florey Neuroscience Institutes, The University of Melbourne, Victoria 3010, Australia.

Relaxin-3 (RLX3), a newly identified member of the relaxin peptide family, is distinguished by its enriched expression in GABA projection neurons of the pontine nucleus incertus (NI), which are postulated to participate in forebrain neural circuits involved in behavioural activation and stress responses. In this regard, corticotrophin-releasing factor-1 receptor (CRF(1)) is abundantly expressed by NI neurons; central CRF administration activates c-fos expression in NI; and various stressors have been reported to increase NI neuron activity. In studies to determine whether a specific neurogenic stressor would activate RLX3 expression, we assessed the effect of a repeated forced swim (RFS) on levels of RLX3 mRNA and heteronuclear (hn) RNA in rat NI by in situ hybridization histochemistry of exon- and intron-directed oligonucleotide probes, respectively. Exposure of rats to an RFS (10 min at 23 degrees C, 24 h apart), markedly increased RLX3 mRNA levels in NI at 30-60 min after the second swim, before a gradual return to basal levels over 2-4 h, while RLX3 hnRNA levels were significantly up-regulated at 60-120 min post-RFS, following a transient decrease at 30 min. Systemic treatment of rats with a CRF(1) antagonist, antalarmin (20 mg/kg, i.p.) 30 min prior to the second swim, blunted the stress-induced effects on RLX3 transcripts. Relative levels of RLX3-immunostaining in NI neurons appeared elevated at 3 h post-swim, but not at earlier time points (30-60 min). These results suggest that acute stress-induced CRF secretion can rapidly alter RLX3 gene transcription by activation of CRF(1) present on NI neurons. More generally, these studies support a role for RLX3 neural networks in the normal neural and physiological response to neurogenic stressors in the rat.
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http://dx.doi.org/10.1016/j.neuropharm.2009.06.019DOI Listing
January 2010

Verification of a relaxin-3 knockout/LacZ reporter mouse as a model of relaxin-3 deficiency.

Ann N Y Acad Sci 2009 Apr;1160:259-60

Howard Florey Institute, University of Melbourne, Victoria, Australia.

Research on the neuropeptide relaxin-3 has predominately been conducted in rats. Anatomical studies have yielded important information on the distribution of relaxin-3 and its cognate receptor (RXFP3) within the brain, while functional studies have implicated relaxin-3 as a modulator of feeding and stress responses. Therefore, the relaxin-3/RXFP3 system represents a potential target for novel drugs to treat human disorders such as obesity, anxiety, and depression, but more research into this interesting neuropeptide in different experimental species is still required. Before conducting detailed neurochemical and behavioral examinations of a recently generated relaxin-3 knockout mouse strain, the present study determined whether this mouse was a viable model of relaxin-3 deficiency.
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http://dx.doi.org/10.1111/j.1749-6632.2009.03952.xDOI Listing
April 2009