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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. [ABSTRACT FROM AUTHOR]

The network formed by the brainstem, cerebellum, and hippocampus occupies a central position to achieve navigation. Multiple physiological functions are implicated in this complex behavior. Among these, control of the eye--head and body movements is crucial. The gaze-holding system realized by the brainstem oculomotor neural integrator (ONI) situated in the nucleus prepositus hypoglossi and fine-tuned by the contribution of different regions of the cerebellum assumes the stability of the image on the fovea. This function helps in the recognition of environmental targets and defining appropriate navigational pathways further elaborated by the entorhinal cortex and hippocampus. In this context, an enigmatic brainstem area situated in front of the ONI, the nucleus incertus (NIC), is implicated in the dynamics of brainstem--hippocampus theta oscillation and contains a group of neurons projecting to the cerebellum. These neurons are characterized by burst tonic behavior similar to the burst tonic neurons in the ONI that convey eye velocity-position signals to the cerebellar flocculus. Faced with these forgotten cerebellar projections of the NIC, the present perspective discusses the possibility that, in addition to the already described pathways linking the cerebellum and the hippocampus via the medial septum, these NIC signals related to the vestibulo-ocular reflex and gaze holding could participate in the hippocampal control of navigation. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Brain-to-brain interfaces (BtBIs) hold exciting potentials for direct communication between individual brains. However, technical challenges often limit their performance in rapid information transfer. Here, we demonstrate an optical brain-to-brain interface that transmits information regarding locomotor speed from one mouse to another and allows precise, real-time control of locomotion across animals with high information transfer rate. We found that the activity of the genetically identified neuromedin B (NMB) neurons within the nucleus incertus (NI) precisely predicts and critically controls locomotor speed. By optically recording Ca2+ signals from the NI of a "Master" mouse and converting them to patterned optogenetic stimulations of the NI of an "Avatar" mouse, the BtBI directed the Avatar mice to closely mimic the locomotion of their Masters with information transfer rate about two orders of magnitude higher than previous BtBIs. These results thus provide proof-of-concept that optical BtBIs can rapidly transmit neural information and control dynamic behaviors across individuals. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The laterodorsal tegmental nucleus (LDTg) is a hindbrain cholinergic cell group thought to be involved in mechanisms of arousal and the control of midbrain dopamine cells. Nowadays, there is increasing evidence that LDTg is also engaged in mechanisms of anxiety/fear and promotion of emotional arousal under adverse conditions. Interestingly, LDTg appears to be connected with other regulators of aversive motivational states, including the lateral habenula (LHb), medial habenula (MHb), interpeduncular nucleus (IP), and median raphe nucleus (MnR). However, the circuitry between these structures has hitherto not been systematically investigated. Here, we placed injections of retrograde or anterograde tracers into LDTg, LHb, IP, and MnR. We also examined the transmitter phenotype of LDTg afferents to IP by combining retrograde tracing with immunofluorescence and in situ hybridization techniques. We found LHb inputs to LDTg mainly emerging from the medial division of the LHb (LHbM), which also receives axonal input from LDTg. The bidirectional connections between IP and LDTg displayed a lateralized organization, with LDTg inputs to IP being predominantly GABAergic or cholinergic and mainly directed to the contralateral IP. Moreover, we disclosed reciprocal LDTg connections with structures involved in the modulation of hippocampal theta rhythm including MnR, nucleus incertus, and supramammillary nucleus. Our findings indicate that the habenula is linked with LDTg either by direct reciprocal projections from/to LHbM or indirectly via the MHb‐IP axis, supporting a functional role of LDTg in the regulation of aversive behaviors, and further characterizing LHb as a master controller of ascending brainstem state‐setting modulatory projection systems. [ABSTRACT FROM AUTHOR]

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 Gi/o‐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. [ABSTRACT FROM AUTHOR]

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 Gi/o-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. [ABSTRACT FROM AUTHOR]

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 Gi/o-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 in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 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. [ABSTRACT FROM AUTHOR]

Alcoholism is a chronic relapsing disorder, and stress is a key precipitant of relapse. The nucleus incertus (NI) is highly responsive to corticotrophin-releasing factor (CRF) and psychological stressors, receives a CRF innervation and expresses CRF1 and CRF2 receptor mRNA. Furthermore, the ascending NI relaxin-3 system is implicated in alcohol seeking in rats. Therefore, in alcohol-preferring rats, we examined the effect of bilateral injections into the NI of the CRF1 receptor antagonist, CP376395 or the CRF2 receptor antagonist, astressin-2B on yohimbine-induced reinstatement of alcohol seeking. Using quantitative PCR analysis of NI micropunches, we assessed the effects of chronic alcohol consumption on gene expression profiles for components of the relaxin-3 and CRF systems. Bilateral intra-NI injections of CP376395 (500 ng/0.25 µl) attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injections of astressin-2B (200 ng/0.25 µl) had no significant effect. In line with these data, CRF1 , but not CRF2 , receptor mRNA was upregulated in the NI following chronic ethanol intake. Relaxin family peptide 3 receptor mRNA was also increased in the NI following chronic ethanol. Our quantitative PCR analysis also identified CRF mRNA within the rat NI, and the existence of a newly identified population of CRF-containing neurons was subsequently confirmed by detection of CRF immunoreactivity in rat and mouse NI. These data suggest that NI neurons contribute to reinstatement of alcohol seeking, via an involvement of CRF1 signalling. Furthermore, chronic ethanol intake leads to neuroadaptive changes in CRF and relaxin-3 systems within rat NI. [ABSTRACT FROM AUTHOR]

The habenula is an epithalamic structure differentiated into two nuclear complexes, medial (MHb) and lateral habenula (LHb). Recently, MHb together with its primary target, the interpeduncular nucleus (IP), have been identified as major players in mediating the aversive effects of nicotine. However, structures downstream of the MHb-IP axis, including the median (MnR) and caudal dorsal raphe nucleus (DRC), may contribute to the behavioral effects of nicotine. The afferent and efferent connections of the IP have hitherto not been systematically investigated with sensitive tracers. Thus, we placed injections of retrograde or anterograde tracers into different IP subdivisions or the MnR and additionally examined the transmitter phenotype of major IP and MnR afferents by combining retrograde tract tracing with immunofluorescence and in situ hybridization techniques. Besides receiving inputs from MHb and also LHb, we found that IP is reciprocally interconnected mainly with midline structures, including the MnR/DRC, nucleus incertus, supramammillary nucleus, septum, and laterodorsal tegmental nucleus. The bidirectional connections between IP and MnR proved to be primarily GABAergic. Regarding a possible topography of IP outputs, all IP subnuclei gave rise to descending projections, whereas major ascending projections, including focal projections to ventral hippocampus, ventrolateral septum, and LHb originated from the dorsocaudal IP. Our findings indicate that IP is closely associated to a distributed network of midline structures that modulate hippocampal theta activity and forms a node linking MHb and LHb with this network, and the hippocampus. Moreover, they support a cardinal role of GABAergic IP/MnR interconnections in the behavioral response to nicotine. [ABSTRACT FROM AUTHOR]

This cover image, by M. Haidar et al., is based on the Research Article Relaxin‐3 inputs target hippocampal interneurons and deletion of hilar relaxin‐3 receptors in “floxed‐RXFP3” mice impairs spatial memory, DOI: 10.1002/hipo.22709. [ABSTRACT FROM AUTHOR]

ABSTRACT Hippocampus is innervated by γ-aminobutyric acid (GABA) 'projection' neurons of the nucleus incertus (NI), including a population expressing the neuropeptide, relaxin-3 (RLN3). In studies aimed at gaining an understanding of the role of RLN3 signaling in hippocampus via its Gi/o-protein-coupled receptor, RXFP3, we examined the distribution of RLN3-immunoreactive nerve fibres and RXFP3 mRNA-positive neurons in relation to hippocampal GABA neuron populations. RLN3-positive elements were detected in close-apposition with a substantial population of somatostatin (SST)- and GABA-immunoreactive neurons, and a smaller population of parvalbumin- and calretinin-immunoreactive neurons in different hippocampal areas, consistent with the relative distribution patterns of RXFP3 mRNA and these marker transcripts. In light of the functional importance of the dentate gyrus (DG) hilus in learning and memory, and our anatomical data, we examined the possible influence of RLN3/RXFP3 signaling in this region on spatial memory. Using viral-based Cre/ LoxP recombination methods and adult mice with a floxed Rxfp3 gene, we deleted Rxfp3 from DG hilar neurons and assessed spatial memory performance and affective behaviors. Following infusions of an AAV(1/2)-Cre-IRES-eGFP vector, Cre expression was observed in DG hilar neurons, including SST-positive cells, and in situ hybridization histochemistry for RXFP3 mRNA confirmed receptor depletion relative to levels in floxed-RXFP3 mice infused with an AAV(1/2)-eGFP (control) vector. RXFP3 depletion within the DG hilus impaired spatial reference memory in an appetitive T-maze task reflected by a reduced percentage of correct choices and increased time to meet criteria, relative to control. In a continuous spontaneous alternation Y-maze task, RXFP3-depleted mice made fewer alternations in the first minute, suggesting impairment of spatial working memory. However, RXFP3-depleted and control mice displayed similar locomotor activity, anxiety-like behavior in light/dark box and elevated-plus maze tests, and learning and long-term memory retention in the Morris water maze. These data indicate endogenous RLN3/RXFP3 signaling can modulate hippocampal-dependent spatial reference and working memory via effects on SST interneurons, and further our knowledge of hippocampal cognitive processing. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The medial septum/diagonal band (MS/DB) is a relay region connecting the hypothalamus and brainstem with the hippocampus, and both the MS/DB and dorsal/ventral hippocampus receive strong topographic GABA/peptidergic projections from the nucleus incertus of the pontine tegmentum. The neuropeptide relaxin-3, released by these neurons, is the cognate ligand for a G-protein-coupled receptor, RXFP3, which is highly expressed within the MS/DB, and both cholinergic and GABAergic neurons in this region of rat brain receive relaxin-3 positive terminals/boutons. Comprehensive in vitro studies have demonstrated that the cell signaling pathways altered by RXFP3 stimulation, include inhibition of forskolin-activated cAMP levels and activation of ERK phosphorylation. In this study we investigated whether intracerebroventricular (icv) injection of RXFP3-A2, a selective relaxin-3 receptor agonist, altered ERK phosphorylation levels in the MS/DB of adult male rats. We subsequently assessed the neurochemical phenotype of phosphorylated (p) ERK-positive neurons in MS/DB after icv RXFP3-A2 administration by dual-label immunostaining for pERK and neuronal markers for cholinergic and GABAergic neurons. Central RXFP3-A2 injection significantly increased levels of pERK immunoreactivity (IR) in MS/DB at 20 and 90 min post-injection, compared to vehicle and naive levels. In addition, RXFP3-A2 increased the number of cells expressing pERK-IR in the MS/DB at 90 (but not 20) min post-injection in cholinergic (but not GABAergic) neurons, which also expressed putative RXFP3-IR. Moreover, icv injection of RXFP3-A2 impaired alternation in a delayed spontaneous T-maze test of spatial working memory. The presence of RXFP3-like IR and the RXFP3-related activation of the MAPK/ERK pathway in MS/DB cholinergic neurons identifies them as a key target of ascending relaxin-3 projections with implications for the acute and chronic modulation of cholinergic neuron activity and function by relaxin-3/RXFP3 signaling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 amygdale-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. [ABSTRACT FROM AUTHOR]

Arousal is a process that involves the activation of ascending neural pathways originating in the rostral pons that project to the forebrain through the midbrain reticular formation to promote the activation of key cortical, thalamic, hypothalamic and limbic centres. Established modulators of arousal include the cholinergic, serotonergic, noradrenergic and dopaminergic networks originating in the pons and midbrain. Recent data indicate that a population of largely GABAergic projection neurones located in the nucleus incertus ( NI) are also involved in arousal and motivational processes. The NI has prominent efferent connections with distinct hypothalamic, amygdalar and thalamic nuclei, in addition to dense projections to key brain regions associated with the generation and pacing of hippocampal activity. The NI receives strong inputs from the prefrontal cortex, lateral habenula and the interpeduncular and median raphe nuclei, suggesting it is highly integrated in circuits regulating higher cognitive behaviours (hippocampal theta rhythm) and emotion. Anatomical and functional studies have revealed that the NI is a rich source of multiple peptide neuromodulators, including relaxin-3, and may mediate extra-hypothalamic effects of the stress hormone corticotrophin-releasing factor, as well as other key modulators such as orexins and oxytocin. This review provides an overview of earlier studies and highlights more recent research that implicates this neural network in the integration of arousal and motivated behaviours and has begun to identify the associated mechanisms. Future research that should help to better clarify the connectivity and function of the NI in major experimental species and humans is also discussed. [ABSTRACT FROM AUTHOR]

ABSTRACT 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. J. Comp. Neurol. 523:565-588, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

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The medial prefrontal cortex ( mPFC) in the rat has been implicated in a variety of cognitive processes, including working memory and expression of fear memory. We investigated the inputs from a brain stem nucleus, the nucleus incertus ( NI), to the prelimbic area of the m PFC. This nucleus strongly expresses corticotropin-releasing factor type 1 ( CRF1) receptors and responds to stress. A retrograde tracer was used to verify connections from the NI to the mPFC. Retrogradely labelled cells in the NI expressed CRF receptors. Electrophysiological manipulation of the NI revealed that stimulation of the NI inhibited spontaneous neuronal firing in the mPFC. Similarly, CRF infusion into the NI, in order to mimic a stressful condition, inhibited neuronal firing and burst firing in the mPFC. The effect of concurrent high-frequency stimulation of the NI on plasticity in the hippocampo-prelimbic medial prefrontal cortical ( HP- mPFC) pathway was studied. It was found that electrical stimulation of the NI impaired long-term potentiation in the HP- mPFC pathway. Furthermore, CRF infusion into the NI produced similar results. These findings might account for some of the extra-pituitary functions of CRF and indicate that the NI may play a role in stress-driven modulation of working memory and possibly other cognitive processes subserved by the m PFC. [ABSTRACT FROM AUTHOR]

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. J. Comp. Neurol. 520:1903-1939, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We report the identification, the cDNA cloning, the temporal and spatial expression pattern analysis of the rln gene in the zebrafish Danio rerio. The deduced Rln B and A domains show different evolutionary conservation. Rln B domain shows higher similarity when compared to zebrafish and human RLN3 B domain than human RLN1 and RLN2 B domain. Differently, the zebrafish Rln A domain shows relatively low amino acid sequence similarity when compared with the same sequences. The rln gene is transcribed both during embryogenesis and in adult organism, where higher transcript level has been particularly evidenced in the brain. Moreover, we provide the first description of rln spatial expression pattern during embryonic development. In particular, we show restricted transcript localization starting at the pharyngula stage in olfactory placode, branchial arch region, and in a cell cluster near to otic vesicle. In larval stage, new transcription territories have been detected in both neural and non-neural regions. In particular, in the brain, rln expression has been revealed in telencephalic region around anterior commissure, in the preoptic area, and in restricted rombencephalic cell clusters. Expression of rln gene in extra-neural territories has been detected in the pancreatic and thyroid gland regions. Danio rerio rln expression pattern analysis reveals shared features with the mammalian RLN gene, particularly in the brain, where it might have a role in the neurophysiological processes. In addition, expression in the thyroid and pancreas region suggests a function as a paracrine and endocrine hormone. [ABSTRACT FROM AUTHOR]

Relaxin-3 (R3) is the latest member of the insulin (INSL) superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide chains. R3 has nearly exclusive expression in the brainstem and was demonstrated to be an additional ligand for LGR7. We recently identified R3 as a ligand for two orphan Gprotein coupled receptors, GPCR135 and GPCR142. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. By contrast, GPCR142 is a pseudogene in the rat, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in the rodent. In addition, the tissue expression pattern of GPCR142, which is primarily in peripheral tissue, is drastically different from that of R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis among INSL/relaxin family members shows that INSL5 is the closest member of R3. We were able to demonstrate that INSL-5 is an agonist for GPCR142 but not for GPCR135. We also showed that the mRNA expression pattern of INSL- 5 overlaps with that of GPCR142. By substituting the A chain of R3 with the A chain of INSL-5, we devised a chimeric peptide (R3/I5) that is about 1000-fold selective for GPCR135 and GPCR142 over LGR7. Autoradiographic distribution of GPCR135 binding sites using [125I]R3/I5 in rat brain shows that GPCR135 receptor is most prominent in areas known for the processing of sensory signals. [ABSTRACT FROM AUTHOR]

G-protein-coupled receptor 135 (GPCR135), a former orphan GPCR also known as SALPR, has recently been shown to be modulated by relaxin-3 (R3). In addition to GPCR135, R3 has been shown to be an agonist for GPCR142 (which is a pseudogene in the rat) and to activate LGR7, which is primarily the receptor for relaxin-1/2. The interaction of R3 with LGR7 has confounded the autoradiographic study of the GPCR135 distribution in the rat CNS due to significant expression of LGR7 in the brain. R3/I5, a chimera of the B-chain of R3 bonded to the A-chain of INSL-5, is a specific GPCR135 agonist which is highly selective for GPCR135 over LGR7. [125I]R3/I5 specifically binds to sites on rat brain sections with a pharmacology matching results from membrane preparations of recombinant GPCR135 receptors. Autoradiographic studies show the GPCR135 receptor density is most prominent in areas such as the olfactory bulb, sensory cortex, amygdala, thalamus, paraventricular nucleus, supraoptic nucleus, inferior and superior colliculus. The GPCR135 mRNA distribution generally overlaps the pattern of GPCR135 binding sites shown by autoradiography using [125I]R3/I5. The nucleus incertus, which has been implicated in the extrapituitary actions of corticotropin-releasing hormone, is the primary source of R3 in the rat central nervous system and expresses GPCR135 receptors. These binding autoradiography and in situ hybridization data suggest that GPCR135 plays an important role in the central processing of sensory signals in rats, are consistent with a putative role for R3/GPCR135 as modulators of stress responses, and confirm the identity of R3 as the central nervous system ligand for GPCR135. Copyright © 2004 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]