Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat

doi:10.1016/j.neuroscience.2006.08.072

Neuroscience

Volume 144, Issue 1, 5 January 2007, Pages 165-190

https://doi.org/10.1016/j.neuroscience.2006.08.072 Get rights and content

Abstract

Relaxin-3 (RLX3) is a newly identified member of the relaxin/insulin peptide family that is highly conserved across a range of species from fish to mammals and is highly expressed in rat, mouse and human brain. Extensive pharmacological studies have demonstrated that RLX3 is a high affinity, selective ligand for G-protein-coupled receptor-135 (GPCR135, now classified as relaxin family peptide-3 receptor; RXFP3). In ongoing studies to understand the physiological functions of RLX3, the distribution of RLX3-containing neuronal elements in rat brain was determined by immunohistochemistry, using an affinity-purified polyclonal antiserum raised against a conserved segment of the RLX3 C-peptide (AS-R3_85-101). Consistent with the distribution of RLX3 mRNA, neurons containing RLX3-like immunoreactivity (LI) were observed in the pontine nucleus incertus and the majority of these cells, which are known to express corticotropin-releasing factor receptor-1, were shown to express glutamic acid decarboxylase-65-immunoreactivity, suggesting a GABA phenotype. Nerve fibers and terminals containing RLX3-LI were observed adjacent to cells in the nucleus incertus and in various forebrain regions known to receive afferents from the nucleus incertus, including cortex, septum, hippocampus, thalamus, hypothalamus and midbrain. Regions that contained highest densities of RLX3-positive fibers included the medial septum, lateral preoptic area, lateral hypothalamus/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and frontal/cingulate cortices, bed nucleus of the stria terminalis, dorsal endopiriform, intergeniculate, and supramammillary nuclei, and the periaqueductal gray and dorsal raphe. The RLX3-positive network overlapped the regional distribution of GPCR135 mRNA and specific binding sites for an [¹²⁵I]-GPCR135-selective, chimeric peptide. These anatomical findings further support the proposition that RLX3 is the endogenous ligand for GPCR135 in rat brain and provide evidence for broad modulatory activity of RLX3 in behavioral activation relating to autonomic and neuroendocrine control of metabolism and reproduction and higher-order processes such as stress and cognition.

Section snippets

Animals

Experiments described were carried out with the approval of the Howard Florey Institute Animal Welfare Committee and according to ethical guidelines issued by the National Health and Medical Research Council of Australia. All efforts were made to minimize the number of animals used and their suffering. Male Sprague–Dawley rats (n=12; 250–300 g) were obtained from the Australian Research Centre (Canning Vale, WA, Australia) and maintained on a 12-h light/dark cycle. Rats were killed by halothane

Specificity of RLX3 antiserum

In the current study, the distribution of RLX3-like immunoreactivity (-LI) was investigated using an affinity-purified polyclonal antiserum raised against a short 16 amino acid sequence of the C-peptide that is identical in the RLX3 sequences of mouse, rat, and human (Bathgate et al 2002, Burazin et al 2002) and the specificity of the immunostaining with AS-R3_85-101 was assessed in various ways. In naïve and colchicine-treated rats, the cellular distribution of RLX3-LI was compatible with that

Discussion

These studies have identified the extensive, topographical distribution of RLX3-LI throughout the adult, male rat brain, with expression detected in specific regions/nuclei of the olfactory bulb, neocortex, limbic forebrain, hypothalamus, thalamus, midbrain, pons and medulla. Highest densities of nerve fibers and terminals were observed in the medial septum, LPO, LH/medial forebrain bundle and ventral hippocampus; and additional fibers were observed in olfactory bulb and olfactory and

Conclusions

The current study provides a clearer understanding of the anatomy of the novel RLX3–GPCR135 signaling system, and provides an excellent foundation for experiments aimed at discovering further details of the neurochemical anatomy of GPCR135-responsive neurons and circuits; and efforts to determine the physiological role(s) of this system in mammalian brain. In this regard, the broad, but distinct neuroanatomical distribution of this system strongly suggests an involvement in behaviors such as

Acknowledgments

This research was supported by grants (983001, 277609 and 300012) from the National Health and Medical Research Council (NHMRC) of Australia, and a collaborative research agreement between the Howard Florey Institute and Johnson & Johnson Pharmaceutical Research & Development, LLC. During these studies S.M. was the recipient of an NHMRC (Australia) Dora Lush Biomedical Postgraduate Scholarship. The authors wish to thank Kelli Johnson for technical assistance with preliminary experiments, Dr.

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The relaxin-3/RXFP3 system is one of several neuropeptidergic systems putatively implicated in regulating the behavioural alterations that characterise clinical depression and anxiety, making it a potential target for clinical translation. Accordingly, this systematic review identified published reports on the role of relaxin-3/RXFP3 signalling in these neuropsychiatric disorders and their behavioural endophenotypes, evaluating evidence from animal and human studies to ascertain any relationship. We searched PubMed, EMBASE, PsycINFO and Google Scholar databases up to February 2021, finding 609 relevant records. After stringent screening, 51 of these studies were included in the final synthesis. There was considerable heterogeneity in study designs and some inconsistency across study outcomes. However, experimental evidence is consistent with an ability of relaxin-3/RXFP3 signalling to promote arousal and suppress depressive- and anxiety-like behaviour. Moreover, meta-analyses of six to eight articles investigating food intake revealed that acute RXFP3 activation had strong orexigenic effects in rats. This appraisal also identified the lack of high-quality clinical studies pertinent to the relaxin-3/RXFP3 system, a gap that future research should attempt to bridge.
Endurance exercise improves avoidance learning and spatial memory, through changes in genes of GABA and relaxin-3, in rats
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Different exercise patterns, neurotransmitters, and some genes have numerous effects on learning and memory. This research aims to investigate the long-term effects of submaximal aerobic exercise on spatial memory (SM), passive avoidance learning (PAL), levels of serum relaxin-3, gamma-aminobutyric acid (GABA), RLN3 gene, and glutamic acid decarboxylase (GAD65/67 genes) in the brainstem of adult male Wistar rats. Fifty male Wistar rats were randomly divided into five groups: aerobic exercise groups, performed on a treadmill running (TR), for 5 weeks (Ex_5, n = 10), 10 weeks (Ex_10, n = 10), involuntary running wheel group for 5 weeks (IRW₅, n = 10), sham (Sh, n = 10) and control (Co, n = 10). Consequently, SM, PAL, serum relaxin-3, GABA, and GAD65/67 and RLN3 genes were measured by ELISA and PCR. Ex_5, Ex₁₀ and IRW₅ improved significantly SM (p ≤ 0.05), PAL (p ≤ 0.001) and decreased significantly relaxin-3 (p ≤ 0.001). RLN3 in the brain also decreased. However, it was not significant. GABA and GAD65/GAD67 increased significantly (p ≤ 0.05) in Ex₅, Ex₁₀ compared to Sh and Co. Aerobic exercise enhanced SM and PAL in Ex compared to Co and Sh. However, duration and type of exercise affected the level of enhancement. The serum relaxin-3 and RLN3 gene displayed reverse functions compared to GABA and GAD65/67 genes in Ex. Therefore, the changes of neurotransmitters in serum relaxin-3, GABA, and their genes: RLN3 and GAD65/67 respectively, influenced learning and memory meaningfully.
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NeuroanatomyRelaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat

Abstract

Section snippets

Animals

Specificity of RLX3 antiserum

Discussion

Conclusions

Acknowledgments

An analysis of the origins of the cholinergic and noncholinergic septal projections to the hippocampal formation of the rat

J Comp Neurol

The connections of the peripeduncular area studied by retrograde and anterograde transport in the rat

J Comp Neurol

Relaxin-3 neurons in the nucleus incertus: Projection patterns, response to swim stress, and RLX3 neuronal signaling

Soc Neurosci Abstr

International union of pharmacology LVII: Recommendations for the nomenclature of receptors for relaxin family peptides

Pharmacol Rev

Relaxin-3: Improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Biochemistry

Relaxin: new peptides, receptors and novel actions

Trends Endocrinol Metab

Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) geneNovel members of the relaxin peptide family

J Biol Chem

A 25 year adventure in the field of tachykinins

Peptides

Conditioned fear-induced changes in behavior and in the expression of the immediate early gene c-fos: with and without diazepam pretreatment

J Neurosci

Do centrally administered neuropeptides access cognate receptors?An analysis in the central corticotropin-releasing factor system

J Neurosci

Restricted, but abundant, expression of the novel rat gene-3 (R3) relaxin in the dorsal tegmental region of brain

J Neurochem

Cholinergic and noncholinergic septal neurons modulate strategy selection in spatial learning

Eur J Neurosci

Distribution of c-Fos in guinea-pig brain following morphine withdrawal

Brain Res

Ontogeny of cholecystokinin-8-containing neuron system of the rat: an immunohistochemical analysisI. Forebrain and upper brainstem

J Comp Neurol

Biochemical and histochemical characterization of ranatensin immunoreactive peptides in rat brain: Lack of coexistence with bombesin/GRP

Brain Res

Studies of the connexions of the fornix system

J Neurochem

Septal GABAergic and hippocampal cholinergic systems modulate anxiety in the plus-maze and shock-probe tests

Pharmacol Biochem Behav

Septal GABAergic and hippocampal cholinergic systems interact in the modulation of anxiety

Neuroscience

C-peptide immunoreactive neurons in human brain

Acta Histochem

Hypothalamic and olfactory control of sexual behavior and partner preference in male rats

Physiol Behav

Appetitive behavior: impact of amygdala-dependent mechanisms of emotional learning

Ann N Y Acad Sci

Peripeduncular nucleus lesions in the rat: IEffects on maternal aggression, lactation, and maternal behavior during pre- and postpartum periods

Behav Neurosci

The thalamic intergeniculate leaflet modulates photoperiod responsiveness in Siberian hamsters

Brain Res

Sleep-related c-Fos protein expression in the preoptic hypothalamus: Effects of ambient warming

Am J Physiol

Projections from the subfornical region of the lateral hypothalamic area

J Comp Neurol

Connections of the nucleus incertus

J Comp Neurol

Opioid-dependent effects of inescapable shock on escape behavior and conditioned fear responding are mediated by the dorsal raphe nucleus

Behav Brain Res

Freewheel running prevents learned helplessness/behavioral depression: Role of dorsal raphe serotonergic neurons

J Neurosci

The role of corticotropin-releasing hormone in the dorsal raphe nucleus in mediating the behavioral consequences of uncontrollable stress

J Neurosci

Food intake, aggression, and fear behavior in the mother rat: Control by neural systems concerned with milk ejection and maternal behavior

Behav Neurosci

The importance of the peripeduncular nucleus in the neuroendocrine control of sexual behavior and milk ejection in the rat

Neuroendocrinology

A parvalbumin-containing, axosomatic synaptic network in the rat medial septum: Relevance to rhythmogenesis

Eur J Neurosci

Mismatches between neurotransmitter and receptor localizations in brain: Observations and implications

Neuroscience

Activity of thermosensitive neurons of monkey preoptic hypothalamus during thermoregulatory operant behavior

Brain Res Bull

Intergeniculate leaflet and ventral lateral geniculate nucleus afferent connections: An anatomical substrate for functional input from the vestibulo-visuomotor system

J Comp Neurol

Activation of orphan receptors by the hormone relaxin

Science

Neuroanatomy
Relaxin-3 in GABA projection neurons of nucleus incertus suggests widespread influence on forebrain circuits via G-protein-coupled receptor-135 in the rat