Unveiling a novel memory center in human brain: neurochemical identification of the nucleus incertus, a key pontine locus implicated in stress and neuropathology.

BACKGROUND: The nucleus incertus (NI) was originally described by Streeter in 1903, as a midline region in the floor of the fourth ventricle of the human brain with an 'unknown' function. More than a century later, the neuroanatomy of the NI has been described in lower vertebrates, but not in humans. Therefore, we examined the neurochemical anatomy of the human NI using markers, including the neuropeptide, relaxin-3 (RLN3), and began to explore the distribution of the NI-related RLN3 innervation of the hippocampus. METHODS: Histochemical staining of serial, coronal sections of control human postmortem pons was conducted to reveal the presence of the NI by detection of immunoreactivity (IR) for the neuronal markers, microtubule-associated protein-2 (MAP2), glutamic acid dehydrogenase (GAD)-65/67 and corticotrophin-releasing hormone receptor 1 (CRHR1), and RLN3, which is highly expressed in NI neurons in diverse species. RLN3 and vesicular GABA transporter 1 (vGAT1) mRNA were detected by fluorescent in situ hybridization. Pons sections containing the NI from an AD case were immunostained for phosphorylated-tau, to explore potential relevance to neurodegenerative diseases. Lastly, sections of the human hippocampus were stained to detect RLN3-IR and somatostatin (SST)-IR. RESULTS: In the dorsal, anterior-medial region of the human pons, neurons containing RLN3- and MAP2-IR, and RLN3/vGAT1 mRNA-positive neurons were observed in an anatomical pattern consistent with that of the NI in other species. GAD65/67- and CRHR1-immunopositive neurons were also detected within this area. Furthermore, RLN3- and AT8-IR were co-localized within NI neurons of an AD subject. Lastly, RLN3-IR was detected in neurons within the CA1, CA2, CA3 and DG areas of the hippocampus, in the absence of RLN3 mRNA. In the DG, RLN3- and SST-IR were co-localized in a small population of neurons. CONCLUSIONS: Aspects of the anatomy of the human NI are shared across species, including a population of stress-responsive, RLN3-expressing neurons and a RLN3 innervation of the hippocampus. Accumulation of phosphorylated-tau in the NI suggests its possible involvement in AD pathology. Further characterization of the neurochemistry of the human NI will increase our understanding of its functional role in health and disease.

Bidirectional Communication between the Pontine Nucleus Incertus and the Medial Septum Is Carried Out by Electrophysiologically-Distinct Neuronal Populations.

Theta oscillations are key brain rhythm involved in memory formation, sensorimotor integration, and control of locomotion and behavioral states. Generation and spatiotemporal synchronization of theta oscillations rely on interactions between brain nuclei forming a large neural network, which includes pontine nucleus incertus (NI). Here we identified distinct populations of NI neurons, based on the relationship of their firing to hippocampal waves, with a special focus on theta oscillations, and the direction and type of interaction with the medial septum (MS) in male, urethane-anesthetized rats. By recording NI neuronal firing and hippocampal LFP, we described NI neurons that fire action potentials in a theta phase-independent or theta phase-locked and delta wave-independent or delta wave-locked manner. Among hippocampal activity-independent NI neurons, irregular, slow-firing, and regular, fast-firing cells were observed, while hippocampal oscillation-/wave-locked NI neurons were of a bursting or nonbursting type. By projection-specific optotagging, we revealed that only fast-firing theta phase-independent NI neurons innervate the MS, rarely receiving feedback information. In contrast, the majority of theta-bursting NI neurons were inhibited by MS stimulation, and this effect was mediated by direct GABAergic input. Described NI neuronal populations differ in reciprocal connections with the septohippocampal system, plausibly forming separate neuronal loops. Our results suggest that theta phase-independent NI neurons participate in theta rhythm generation through direct innervation of the MS, while theta-bursting NI neurons further transmit the rhythmic signal received from the MS to stabilize and/or strengthen rhythmic activity in other structures.SIGNIFICANCE STATEMENT The generation and spatiotemporal synchronization of theta oscillations rely on interactions between nuclei forming a large neural network, part of which is the pontine nucleus incertus (NI). Here we describe that within NI there are populations of neurons that can be distinguished based on the relationship of their firing to hippocampal theta oscillations and delta waves. We show that these neuronal populations largely do not have reciprocal connections with the septohippocampal system, but form separate neuronal loops. Our results suggest that medial septum (MS)-projecting, fast-firing, theta phase-independent NI neurons may participate in theta rhythm generation through direct innervation of the MS, while theta-bursting NI neurons may further transmit the rhythmic signal received from the MS to other structures.

Levels of spexin and its receptors GALR2 and GALR3 in the hypothalamus and ovary of letrozole-induced polycystic ovary syndrome in rats.

Spexin (SPX) is a newly identified neuropeptide, a natural ligand for the galanin receptors (GALR) 2/3, which is involved in maintaining physiological functions including female reproduction. One of the most common endocrine disorder in reproductive system is polycystic ovary syndrome (PCOS), however the role of SPX in PCOS is still unknown. The objective of this study was to determine the expression of mRNA and peptide levels of SPX and its receptors GALR2/3 in the hypothalamus and ovary (by real time PCR and Western blot) as well as plasma levels of SPX (ELISA) in letrozole - induced PCOS rats. We observed that SPX plasma level does not change in PCOS rats. In the hypothalamus transcript level of Spx and Galr3 were significantly higher in PCOS rats compared to the control, while mRNA of Galr2 and protein expression of GALR2/3 were lower. Moreover, expression of Spx and Galr2/3 mRNA as well as GALR2/3 peptide production were lower in the ovary of PCOS rats. In summary, while our results did not show differences in plasma SPX levels, we observed tissue-dependent significant differences in the SPX/GALR2/3 levels between PCOS and control rats, what indicates possible new mechanisms of PCOS neuroendocrinology.

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.

Altered vitamin D3 metabolism in the ovary and periovarian adipose tissue of rats with letrozole-induced PCOS.

Vitamin D3 (VD3) plays an important role in the ovary and its deficiency is associated with ovarian pathologies, including polycystic ovary syndrome (PCOS). However, there is no data related to VD3 metabolism in the ovary during PCOS. Herein, we investigated differences in the expression of VD3 receptor (VDR) and key VD3 metabolic enzymes, 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), in the ovary and periovarian adipose tissue (POAT) of control (proestrus and diestrus) and PCOS induced by letrozole rats. Vdr, Cyp27b1 and Cyp24a1 mRNA expression was determined, their protein abundance was examined and immunolocalized. Furthermore, VD3 metabolite concentrations in plasma (25OHD) and tissues (ovary and POAT; 1,25(OH)2D3), and plasma calcium level were determined. 25OHD concentration decreased markedly in letrozole-treated rats in comparison with controls, whereas calcium concentration did not vary among the examined groups. The amount of 1,25(OH)2D3 decreased in both ovary and POAT of PCOS rats. In the ovary, we found decreased Cyp27b1 and increased Vdr mRNA expression in letrozole-treated and diestrus control group. Corresponding protein abundances were down-regulated and up-regulated, respectively but only following letrozole treatment. In POAT, only Cyp27b1 transcript level and CYP27B1 protein abundance were decreased in letrozole-treated rats. VDR was immunolocalized in healthy and cystic follicles, while CYP27B1 and CYP24A1 were found exclusively in healthy ones. Concluding, our results provide the first evidence of disrupted VD3 metabolism in the ovary and POAT of PCOS rats. The reduced 1,25(OH)2D3 concentration in those tissues suggests their contribution to VD3 deficiency observed in PCOS and might implicate in PCOS pathogenesis.

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.

Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome.

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology.

Inhibition of oxytocin and vasopressin neuron activity in rat hypothalamic paraventricular nucleus by relaxin-3-RXFP3 signalling.

KEY POINTS: Relaxin-3 is a stress-responsive neuropeptide that acts at its cognate receptor, RXFP3, to alter behaviours including feeding. In this study, we have demonstrated a direct, RXFP3-dependent, inhibitory action of relaxin-3 on oxytocin and vasopressin paraventricular nucleus (PVN) neuron electrical activity, a putative cellular mechanism of orexigenic actions of relaxin-3. We observed a Gαi/o -protein-dependent inhibitory influence of selective RXFP3 activation on PVN neuronal activity in vitro and demonstrated a direct action of RXFP3 activation on oxytocin and vasopressin PVN neurons, confirmed by their abundant expression of RXFP3 mRNA. Moreover, we demonstrated that RXFP3 activation induces a cadmium-sensitive outward current, which indicates the involvement of a characteristic magnocellular neuron outward potassium current. Furthermore, we identified an abundance of relaxin-3-immunoreactive axons/fibres originating from the nucleus incertus in close proximity to the PVN, but associated with sparse relaxin-3-containing fibres/terminals within the PVN. ABSTRACT: The paraventricular nucleus of the hypothalamus (PVN) plays an essential role in the control of food intake and energy expenditure by integrating multiple neural and humoral inputs. Recent studies have demonstrated that intracerebroventricular and intra-PVN injections of the neuropeptide relaxin-3 or selective relaxin-3 receptor (RXFP3) agonists produce robust feeding in satiated rats, but the cellular and molecular mechanisms of action associated with these orexigenic effects have not been identified. In the present studies, using rat brain slices, we demonstrated that relaxin-3, acting through its cognate G-protein-coupled receptor, RXFP3, hyperpolarized a majority of putative magnocellular PVN neurons (88%, 22/25), including cells producing the anorexigenic neuropeptides, oxytocin and vasopressin. Importantly, the action of relaxin-3 persisted in the presence of tetrodotoxin and glutamate/GABA receptor antagonists, indicating its direct action on PVN neurons. Similar inhibitory effects on PVN oxytocin and vasopressin neurons were produced by the RXFP3 agonist, RXFP3-A2 (82%, 80/98 cells). In situ hybridization histochemistry revealed a strong colocalization of RXFP3 mRNA with oxytocin and vasopressin immunoreactivity in rat PVN neurons. A smaller percentage of putative parvocellular PVN neurons was sensitive to RXFP3-A2 (40%, 16/40 cells). These data, along with a demonstration of abundant peri-PVN and sparse intra-PVN relaxin-3-immunoreactive nerve fibres, originating from the nucleus incertus, the major source of relaxin-3 neurons, identify a strong inhibitory influence of relaxin-3-RXFP3 signalling on the electrical activity of PVN oxytocin and vasopressin neurons, consistent with the orexigenic effect of RXFP3 activation observed in vivo.

Diverse action of repeated corticosterone treatment on synaptic transmission, neuronal plasticity, and morphology in superficial and deep layers of the rat motor cortex.

One of the adverse effects of prolonged stress in rats is impaired performance of skilled reaching and walking tasks. The mechanisms that lead to these abnormalities are incompletely understood. Therefore, we compared the effects of twice daily repeated corticosterone injections for 7 days on miniature excitatory postsynaptic currents (mEPSCs), as well as on synaptic plasticity and morphology of layers II/III and V pyramidal neurons of the primary motor cortex (M1) of male Wistar rats. Corticosterone treatment resulted in increased frequency, but not amplitude, of mEPSCs in layer II/III neurons accompanied by increased complexity of the apical part of their dendritic tree, with no changes in the density of dendritic spines. The frequency and amplitude of mEPSCs as well as the parameters characterizing the complexity of the dendritic tree were not changed in layer V cells; however, their dendritic spine density was increased. While corticosterone treatment resulted in an increase in the amplitude of field potentials evoked in intralaminar connections within layer II/III, it did not influence field responses in layer V intralaminar connections, as well as the extent of chemically induced layer V long-term potentiation (chemLTP) by the application of tetraethylammonium (TEA, 25 mM). However, chemLTP induction in layer II/III was impaired in slices prepared from corticosterone-treated animals. These data indicate that repeated 7-day administration of exogenous corticosterone induces structural and functional plasticity in the M1, which occurs mainly in layer II/III pyramidal neurons. These findings shed light on potential sites of action and mechanisms underlying stress-induced impairment of motor functions.

Short-term repeated corticosterone administration enhances glutamatergic but not GABAergic transmission in the rat motor cortex.

It has been demonstrated that stress impairs performance of skilled reaching and walking tasks in rats due to the action of glucocorticoids involved in the stress response. Skilled reaching and walking are controlled by the primary motor cortex (M1); however, it is not known whether stress-related impairments in skilled motor tasks are related to functional and/or structural alterations within the M1. We studied the effects of single and repeated injections of corticosterone (twice daily for 7 days) on spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs) recorded from layer II/III pyramidal neurons in ex vivo slices of the M1, prepared 2 days after the last administration of the hormone. We also measured the density of dendritic spines on pyramidal cells and the protein levels of selected subunits of AMPA, NMDA, and GABAA receptors after repeated corticosterone administration. Repeatedly administered corticosterone induced an increase in the frequency but not in the amplitude of sEPSCs, while a single administration had no effect on the recorded excitatory currents. The frequency and amplitude of sIPSCs as well as the excitability of pyramidal cells were changed neither after single nor after repeated corticosterone administration. Treatment with corticosterone for 7 days did not modify the density of dendritic spines on pyramidal neurons. Corticosterone influenced neither the protein levels of GluA1, GluA2, GluN1, GluN2A, and GluN2B subunits of glutamate receptors nor those of α1, ß2, and γ2 subunits of the GABAA receptor. The increase in sEPSCs frequency induced by repeated corticosterone administration faded out within 7 days. These data indicate that prolonged administration of exogenous corticosterone selectively and reversibly enhances glutamatergic, but not GABAergic transmission in the rat motor cortex. Our results suggest that corticosterone treatment results in an enhancement of spontaneous glutamate release from presynaptic terminals in the M1 and thereby uncovers a potential mechanism underlying stress-induced motor functions impairment.

[Relaxin-3 and relaxin family peptide receptors--from structure to functions of a newly discovered mammalian brain system]. / Relaksyna-3 i receptory rodziny peptydów relaksynowych--od struktury po funkcje nowo odkrytego ukladu mózgowia ssaków.

Relaxin-3, a member of the relaxin peptide family, was discovered in 2001 as a homologue of relaxin--a well-known reproductive hormone. However, it is the brain which turned out to be a major expression site of this newly discovered peptide. Both its molecular structure and expression pattern were shown to be very conserved among vertebrates. Extensive research carried out since the discovery of relaxin-3 contributed to the significant progress in our knowledge regarding this neuropeptide. The endogenous relaxin-3 receptor (RXFP3) was identified and the anatomy of the yet uncharacterized mammalian brain system was described, with nucleus incertus as the main center of relaxin-3 expression. Not only its diffusive projections throughout the whole brain, which reach various brain structures such as the hippocampus, septum, intergeniculate leaflet or amygdala, but also functional studies of the relaxin-3/RXFP3 signaling system, allowed this brain network to be classified as one of the ascending nonspecific brain systems. Thus far, research depicts the connection of relaxin-3 with phenomena such as feeding behavior, spatial memory, sleep/wake cycle or modulation of pituitary gland hormone secretion. Responsiveness of relaxin-3 neurons to stress factors and the strong orexigenic effect exerted by this peptide suggest its participation in modulation of feeding by stress, in particular of the chronic type. The discovery of relaxin-3 opened a new research field which will contribute to our better understanding of the neurobiological basis of feeding disorders.

Dopamine Receptor-Expressing Neurons Are Differently Distributed throughout Layers of the Motor Cortex to Control Dexterity.

The motor cortex comprises the primary descending circuits for flexible control of voluntary movements and is critically involved in motor skill learning. Motor skill learning is impaired in patients with Parkinson's disease, but the precise mechanisms of motor control and skill learning are still not well understood. Here we have used transgenic mice, electrophysiology, in situ hybridization, and neural tract-tracing methods to target genetically defined cell types expressing D1 and D2 dopamine receptors in the motor cortex. We observed that putative D1 and D2 dopamine receptor-expressing neurons (D1+ and D2+, respectively) are organized in highly segregated, nonoverlapping populations. Moreover, based on ex vivo patch-clamp recordings, we showed that D1+ and D2+ cells have distinct morphological and electrophysiological properties. Finally, we observed that chemogenetic inhibition of D2+, but not D1+, neurons disrupts skilled forelimb reaching in adult mice. Overall, these results demonstrate that dopamine receptor-expressing cells in the motor cortex are highly segregated and play a specialized role in manual dexterity.

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

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.

Relaxin-3 innervation of the intergeniculate leaflet of the rat thalamus - neuronal tract-tracing and in vitro electrophysiological studies.

Behavioural state is controlled by a range of neural systems that are sensitive to internal and external stimuli. The relaxin-3 and relaxin family peptide receptor 3 (RXFP3) system has emerged as a putative ascending arousal network with putative involvement in regulation of stress responses, neuroendocrine control, feeding and metabolism, circadian activity and cognition. Relaxin-3/γ-aminobutyric acid neuron populations have been identified in the nucleus incertus, pontine raphe nucleus, periaqueductal grey (PAG) and an area dorsal to the substantia nigra. Relaxin-3-positive fibres/terminals densely innervate arousal-related structures in the brainstem, hypothalamus and limbic forebrain, but the functional significance of the heterogeneous relaxin-3 neuron distribution and its inputs to specific brain areas are unclear. Therefore, in this study, we used neuronal tract-tracing and immunofluorescence staining to explore the source of the dense relaxin-3 innervation of the intergeniculate leaflet (IGL) of the thalamus, a component of the neural circadian timing system. Confocal microscopy analysis revealed that relaxin-3-positive neurons retrogradely labelled from the IGL were predominantly present in the PAG and these neurons expressed corticotropin-releasing factor receptor-like immunoreactivity. Subsequently, whole-cell patch-clamp recordings revealed heterogeneous effects of RXFP3 activation in the IGL by the RXFP3 agonist, relaxin-3 B-chain/insulin-like peptide-5 A-chain (R3/I5). Identified, neuropeptide Y-positive IGL neurons, known to influence suprachiasmatic nucleus activity, were excited by R3/I5, whereas neurons of unidentified neurotransmitter content were either depolarized or displayed a decrease in action potential firing and/or membrane potential hyperpolarization. Our data identify a PAG to IGL relaxin-3/RXFP3 pathway that might convey stress-related information to key elements of the circadian system and influence behavioural state rhythmicity.

Effect of Vitamin D3 on Chemerin and Adiponectin Levels in Uterus of Polycystic Ovary Syndrome Rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder with disrupted uterus structure and function. A positive effect of vitamin D3 (VD3) in female reproduction was observed. Chemerin (RARRES2) and adiponectin (ADIPOQ) are the main adipokines whose levels are altered in PCOS patients. Therefore, the aim of this study was to investigate the impact of VD3 supplementation on RARRES2 and ADIPOQ levels in the uterus of PCOS rats. METHODS: We analyzed the plasma levels and uterine transcript and protein expression of RARRES2 and ADIPOQ and their receptors (CCRL2, CMKLR1, GPR1, and ADIPOR1 and ADIPOR2, respectively) in rats with letrozole-induced PCOS. RESULTS: In control animals, VD3 did not change plasma levels of both adipokines, while in PCOS rats supplemented with VD3, they returned to control levels. The expression of RARRES2 and all investigated receptors increased in the uterus of VD3-treated rats; however, the levels of Rarres2 and Gpr1 genes remained unchanged. VD3 supplementation decreased RARRES2, CMKLR1, and GPR1 but increased CCRL2 level to the control value. In the uterus of VD3-treated rats, the transcript and protein levels of ADIPOQ and both receptors ADIPOR1 increased. At the same time, VD3 supplementation induced an increase in Adipoq, Adipor1, and Adipor2 gene expression and restored protein levels to control level values. CONCLUSIONS: our findings indicate a new mechanism of VD3 action in the uterine physiology of PCOS rats.

An analgesic pathway from parvocellular oxytocin neurons to the periaqueductal gray in rats.

The hypothalamic neuropeptide oxytocin (OT) exerts prominent analgesic effects via central and peripheral action. However, the precise analgesic pathways recruited by OT are largely elusive. Here we discovered a subset of OT neurons whose projections preferentially terminate on OT receptor (OTR)-expressing neurons in the ventrolateral periaqueductal gray (vlPAG). Using a newly generated line of transgenic rats (OTR-IRES-Cre), we determined that most of the vlPAG OTR expressing cells targeted by OT projections are GABAergic. Ex vivo stimulation of parvocellular OT axons in the vlPAG induced local OT release, as measured with OT sensor GRAB. In vivo, optogenetically-evoked axonal OT release in the vlPAG of as well as chemogenetic activation of OTR vlPAG neurons resulted in a long-lasting increase of vlPAG neuronal activity. This lead to an indirect suppression of sensory neuron activity in the spinal cord and strong analgesia in both female and male rats. Altogether, we describe an OT-vlPAG-spinal cord circuit that is critical for analgesia in both inflammatory and neuropathic pain models.

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.

Early-life Stress Modifies the Reactivity of Neurons in the Ventral Tegmental Area and Lateral Hypothalamus to Acute Stress in Female Rats.

Early-life stress (ELS) has long-term consequences, including an increased risk for drug abuse and psychiatric disorders later in life, which is higher in women than in men. The consequences of ELS include heightened sensitivity to stressful events. Here, we hypothesized that ELS changes the stress sensitivity of dopaminergic (DAergic) neurons in the ventral tegmental area (VTA) and orexin (OXA) neurons in the lateral hypothalamus (LH), that are crucial for the control of motivated behaviors. We combined morphological and immunohistochemical approaches to investigate the effect of maternal separation (MS), a rodent model of the ELS, on the expression of c-Fos protein in putative DAergic and non-DAergic VTA and LH OXA neurons, as well as on dendritic spine density and morphology in the VTA of adult female rats. We showed that MS increased basal and acute restraint stress-induced c-Fos expression in putative DAergic neurons, specifically in the dorsomedial VTA, an area implicated in responsiveness to aversive stimuli. Conversely, restraint-induced increase in c-Fos expression in non-DAergic dorsolateral VTA neurons was dampened by MS. Furthermore, an increase in spine head diameter of VTA neurons and a concurrent decrease in dendritic spine density in dorsal VTA were observed. We also showed that MS changed the stress sensitivity of OXA neurons selectively in the dorsomedial hypothalamus (DMH), which is implicated in arousal and the stress response. These findings show the long-lasting consequences of ELS and indicate the selective, regional sensitivity of structures involved in the control of arousal, motivational behaviors and the stress response to ELS.

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.