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.

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.

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.

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.

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.

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

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

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

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

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

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

Early life stress-induced alterations in the activity and morphology of ventral tegmental area neurons in female rats.

Childhood maltreatment, which can take the form of physical or psychological abuse, is experienced by more than a quarter of all children. Early life stress has substantial and long-term consequences, including an increased risk of drug abuse and psychiatric disorders in adolescence and adulthood, and this risk is higher in women than in men. The neuronal mechanisms underlying the influence of early life adversities on brain functioning remain poorly understood; therefore, in the current study, we used maternal separation (MS), a rodent model of early-life neglect, to verify its influence on the properties of neurons in the ventral tegmental area (VTA), a brain area critically involved in reward and motivation processing. Using whole-cell patch-clamp recordings in brain slices from adolescent female Sprague-Dawley rats, we found an MS-induced increase in the excitability of putative dopaminergic (DAergic) neurons selectively in the medial part of the VTA. We also showed an enhancement of excitatory synaptic transmission in VTA putative DAergic neurons. MS-induced alterations in electrophysiology were accompanied by an increase in the diameter of dendritic spine heads on lateral VTA DAergic neurons, although the overall dendritic spine density remained unchanged. Finally, we reported MS-related increases in basal plasma ACTH and corticosterone levels. These results show the long-term consequences of early life stress and indicate the possible neuronal mechanisms of behavioral disturbances in individuals who experience early life neglect.

Electrophysiology and distribution of oxytocin and vasopressin neurons in the hypothalamic paraventricular nucleus: a study in male and female rats.

Magnocellular neurosecretory cells (MNCs) clustered in the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus constitute a major source of oxytocin (OXT) and arginine vasopressin (AVP) peptides, and are among the best described peptidergic neurons in the brain. OXT and AVP are involved in a range of homeostatic processes, social behaviours, emotional processes, and learning. Notably, their actions can be sex-specific, and several sex differences in the anatomies of the OXT and AVP systems have been reported. Nonetheless, possible sex differences in the detailed distributions of MNCs and in their intrinsic electrical properties ex vivo have not been extensively examined. We addressed these issues utilizing immunostaining and patch-clamp ex vivo recordings. Here, we showed that Sprague-Dawley rat PVN AVP neurons are more numerous than OXT cells and that more neurons of both types are present in males. Furthermore, we identified several previously unreported differences between putative OXT and AVP MNC electrophysiology contributing to their partially unique profiles. Notably, elucidation of the highly specific action potential (AP) shapes, with AVP MNCs having a narrower AP and faster hyperpolarizing after-potential (HAP) kinetics than OXT MNCs, allowed unambiguous discrimination between OXT and AVP MNCs ex vivo for the first time. Moreover, the examined electrophysiological properties of male and female MNCs generally overlapped with the following exceptions: higher membrane resistance in male MNCs and HAP kinetics in putative OXT MNCs, which was slower in males. These reported observations constitute a thorough addition to the knowledge of MNC properties shaping their diverse physiological actions in both sexes.

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

The rat nucleus incertus (NI) contains GABA/peptide-projection neurons responsive to orexin (hypocretin)/orexin receptor-2 (OX2) signalling. Melanin-concentrating hormone (MCH) and orexin neurons often innervate and influence common target areas. Therefore, we assessed the relationship between these hypothalamic peptidergic systems and rat NI, by investigating the presence of an MCH innervation and MCH receptor-1 (MCH1) expression, and neurophysiological and behavioural effects of MCH c.f. orexin-A (OXA), within the NI. We identified lateral hypothalamus (LH), perifornical and sub-zona incerta MCH neurons that innervate NI, and characterised the rostrocaudal distribution of MCH-containing fibres in NI. Single-cell RT-PCR detected MCH1 and OX2 mRNA in NI, and multiplex, fluorescent in situ hybridisation revealed distinct co-expression patterns of MCH1 and OX2 mRNA in NI neurons expressing vesicular GABA transporter (vGAT) mRNA. Patch-clamp recordings revealed 34% of NI neurons tested were hyperpolarised by MCH (1 µM), representing a distinct population from OXA-sensitive NI neurons (35%). Intra-NI OXA infusion (600 pmol) in satiated rats during the light/inactive phase produced increased locomotor activity and food (standard chow) intake, whereas intra-NI MCH infusion (600 pmol) produced only a trend for decreased locomotor activity and no effect on food intake. Furthermore, in satiated or pre-fasted rats tested during the dark/active phase, intra-NI infusion of MCH did not alter the elevated locomotor activity or higher food intake observed. However, quantification of neuropeptide-immunostaining revealed differential diurnal fluctuations in orexin and MCH trafficking to NI. Our findings identify MCH and orexin inputs onto divergent NI populations which may differentially influence arousal and motivated behaviours.