Neuropeptide W Research Articles

Neuropeptide B Induces Slow Wave Sleep in Mice

Neuropeptide B (NPB) and neuropeptide W (NPW) are two recently identified neuropeptides that act as endogenous ligands to orphan G protein coupled receptors, GPR7 and GPR8. In rodents, the GPR8 ortholog is absent and both NPB and NPW function exclusively through GPR7. Although NPB and NPW are implicated in the regulation of feeding behavior, endocrine function, and pain sensation, their physiological role is incompletely understood. NPB or saline was administered into the lateral ventricle of mice during both the light and dark periods. In separate experiments, spontaneous locomotor activity or EEG and EMG were recorded after intracerebroventricular (i.c.v). injection. To confirm the involvement of GPR7 in NPB-induced responses, GPR7 knockout mice were also subjected to i.c.v. injections. NPB injections reduced locomotor activity during the dark period, but not during the light period. EEG and EMG recordings in freely moving mice revealed that NPB injection decreased the time spent in the waking state and increased the time spent in slow wave sleep (SWS) during the dark period. The time spent in paradoxical sleep was unaffected. The spectral power of NPB-induced SWS was indistinguishable from that of physiological SWS. The NPB-induced increase in SWS was not observed in GPR7 knockout mice. These results suggest that NPB induced physiological SWS through GPR7 and that NPB and GPR7 may have a role in modulating the occurrence of sleep and wakefulness.

Analgesic Neuropeptide W Suppresses Seizures in the Brain Revealed by Rational Repositioning and Peptide Engineering

Anticonvulsant neuropeptides play an important role in controlling neuronal excitability that leads to pain or seizures. Based on overlapping inhibitory mechanisms, many anticonvulsant compounds have been found to exhibit both analgesic and antiepileptic activities. An analgesic neuropeptide W (NPW) targets recently deorphanized G-protein coupled receptors. Here, we tested the hypothesis that the analgesic activity of NPW may lead to the discovery of its antiepileptic properties. Indeed, direct administration of NPW into the brain potently reduced seizures in mice. To confirm this discovery, we rationally designed, synthesized, and characterized NPW analogues that exhibited anticonvulsant activities following systemic administration. Our results suggest that the combination of neuropeptide repositioning and engineering NPW analogues that penetrate the blood-brain barrier could provide new drug leads, not only for the treatment of epilepsy and pain but also for studying effects of this peptide on regulating feeding and energy metabolism coupled to leptin levels in the brain.

Association of neuropeptide W, but not obestatin, with energy intake and endocrine status in Zucker rats. A new player in long-term stress-feeding interactions

The aim of this study was to ascertain the roles of neuropeptide W (NPW) and obestatin in feeding and endocrine regulations and their interactions with leptin, corticosterone, and insulin, three key hormones involved in metabolic homeostasis. Plasma variations were measured in obese hyperphagic Zucker rats either following a one-day fast, or after chronic food restriction (one-third less food than normal for three weeks). Obestatin did not vary by feeding condition, and did not differ between lean and obese rats; it likely does not play any role in feeding regulation. NPW did not vary with one-day fasting, but was higher in obese rats than in lean rats under satiated (+38%) and fasting (+44%; P<0.01) conditions. In chronically food-restricted obese rats that lost about 10% of their initial body weight, NPW decreased by 18% (P<0.02), in parallel with a similar decrease in plasma insulin (P<0.03), and a 10% decrease of plasma leptin (P<0.001). Corticosterone levels in obese rats were much higher than in lean rats, and increased (P<0.0001) after chronic food restriction, but not after a short fast. Prolonged food restriction was therefore stressful for obese rats. Long-term food shortage associated with insulin, leptin and corticosterone changes is then a critical factor for the regulation of NPW. The NPW up-regulation in hyperphagic conditions and its down-regulation in hypophagic conditions, is compatible with an anorexigenic role of this peptide. NPW thus may be one of the regulatory factors involved in the complex long-term interactions between stress and feeding.

Neuropeptide W: a key player in the homeostatic regulation of feeding and energy metabolism?

Neuropeptide W (NPW), recently isolated from porcine hypothalamus, has been identified as the endogenous ligand for both NPBWR1 (GPR7) and NPBWR2 (GPR8), which belong to the orphan G protein-coupled receptor family. NPW is thought to play an important role in the regulation of feeding and drinking behavior, and to be related to the stress response. NPW-containing neurons are localized in several regions of the brain, including the hypothalamus, hippocampus, limbic system, midbrain, and brain stem. Accumulated evidence suggests that hypothalamic neuropeptides, such as neuropeptide Y (NPY), orexin, melanin-concentrating hormone (MCH), and proopiomelanocortin (POMC), are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. NPW also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. We summarize our current understanding of the distribution of NPW and of the neuronal interactions between NPW and the different feeding-regulating peptide-containing neurons. This review also discusses evidence for the dichotomous actions of NPW on energy balance and the potential mechanisms involved.

Behavioral Actions of Neuropeptide W: More Evidence for a Role in the Stress Response

Neuropeptide W (NPW), an endogenous peptide ligand for GPR7 and GPR8, is produced in hypothalamic and brain stem regions important in the neuroendocrine, behavioral and autonomic responses to stress. Indeed, we demonstrated that central administration of NPW activated the hypothalamo‐pituitary adrenal axis (HPA). We then determined if NPW could alter locomotor activity or mean arterial pressure (MAP), two additional markers of the stress response. Lateral cerebroventricle (i.c.v.) cannulae were implanted in adult male rats 5 days before experimentation. In behavioral testing, basal activity was monitored for 1 hr prior to the administration of saline, 0.1, 0.3, 1, or 3 nmol NPW23 i.c.v. and behavior was monitored for 2 hr. For cardiovascular experiments, a cannula was implanted into the carotid artery. On the following day after recording baseline MAP, rats were injected with 1 nmol NPW23 i.c.v., and MAP was recorded for 1 hr. NPW treatment increased total locomotor activity in a dose‐related manner, and increased stereotypy and ambulatory activity, but did not significantly alter MAP, indicating NPW alters endocrine and behavioral responses to stress without inducing the cardiovascular effects.

Neuropeptide W: An Anorectic Peptide Regulated by Leptin and Metabolic State

Neuropeptide W (NPW) is an anorectic peptide produced in the brain. Here, we showed that NPW was present in several hypothalamic nuclei, including the paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, lateral hypothalamus, and hypothalamic arcuate nucleus. NPW expression was significantly up-regulated in leptin-deficient ob/ob and leptin receptor-deficient db/db mice. The increase in NPW expression in ob/ob mice was abrogated to control levels after leptin replacement. Leptin induced suppressors of cytokine signaling-3 after phosphorylation of signal transducer and activator of transcription-3 in NPW-expressing neurons. In addition, we demonstrated that NPW reduces feeding via the melanocortin-4-receptor signaling pathway. We also showed that NPW activates proopiomelanocortin and inhibits neuropeptide Y neurons using loose-patch extracellular recording of these neurons identified by promoter-driven green fluorescent protein expression. This study indicates that NPW may play an important role in the regulation of feeding and energy metabolism under the conditions of leptin insufficiency.

Feeding regulation and energy metabolism in the brain

Event Abstract Back to Event Feeding regulation and energy metabolism in the brain Seiji Shioda1*, Fumiko Takenoya1, Kanako Shiba1 and Haruaki Kageyama1 1 Showa University School of Medicine, Department of Anatomy, Japan Novel neuropeptides of G-protein coupled receptor (GPCR) ligands are shown to be localized in brain and play a range of physiological functions including feeding regulation and energy metabolism. Here, I will describe the distribution and localization of these new GPCR ligands identified recently and to review their involvement in neuronal networks, particularly feeding regulation and energy metabolism in the brain. This presentation concerns some novel GPCR ligands of feeding and energy metabolismregulating neuropeptides such as orexin, ghrelin, galanin-like peptide (GALP) and neuropeptide W (NPW), such as those studied by our group and other collaborating groups, and neuronal interactions among these and other well known neuropeptides such as neuropeptide Y (NPY) and alpha-melanocyte stimulating hormone (alpha-MSH) in the hypothalamus. Cross-talk among several of these neuropeptides-containing neurons in the hypothalamus plays a crucial role in determining feeding states as well as feeding behavior. I will show some structural and functional characteristics of these recently discovered neuropeptides and summarize the known interactions between these different kind of feeding regulating neurons and leptin-targeting neurons in the hypothalamus. Moreover, I will show a new strategy for analyzing the neural circuit of these feeding- and energy metabolism-regulating GPCR ligands-containing neurons in the brain by use of transgenic model mice. Finally, I will present our recent results of GALP which are involved in regulation of feeding as well as energy homeostasis and body temperature. In addition, I will show our hot data of intranasal infusion of GALP to decrease body weight and locomotor activity in normal and obese mice. Research in this field will serve a very important role of clarifying neurologically-based causes for appetite dysfunctions and life-style diseases and it may help to establish and to lead new therapies for people and/or patients who are suffering from such conditions. Keywords: comparative endocrinology Conference: 25th Conference of the European Comparative Endocrinologists, Pécs, Hungary, 31 Aug - 4 Sep, 2010. Presentation Type: Conference Presentation Topic: Comparative endocrinology Citation: Shioda S, Takenoya F, Shiba K and Kageyama H (2010). Feeding regulation and energy metabolism in the brain. Front. Endocrinol. Conference Abstract: 25th Conference of the European Comparative Endocrinologists. doi: 10.3389/conf.fendo.2010.01.00051 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Aug 2010; Published Online: 29 Aug 2010. * Correspondence: Dr. Seiji Shioda, Showa University School of Medicine, Department of Anatomy, Tokyo, Japan, shioda@hoshi.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Seiji Shioda Fumiko Takenoya Kanako Shiba Haruaki Kageyama Google Seiji Shioda Fumiko Takenoya Kanako Shiba Haruaki Kageyama Google Scholar Seiji Shioda Fumiko Takenoya Kanako Shiba Haruaki Kageyama PubMed Seiji Shioda Fumiko Takenoya Kanako Shiba Haruaki Kageyama Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Distribution of neuropeptide W in the rat brain

Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion.

Neuropeptide B (NPB) and neuropeptide W (NPW) system in cultured rat calvarial osteoblast-like (ROB) cells: NPW and NPB inhibit proliferative activity of ROB cells

Neuropeptides B (NPB) and W (NPW) have been identified as endogenous ligands of two G-protein-coupled receptors, neuropeptides B/W receptor 1 (NPBWR1, formerly known as GPR7) and neuropeptides B/W receptor 2 (NPBWR2, formerly known as GPR8). In rodents where NPBWR2 is absent, its counterpart is named the similar to neuropeptides B/W receptor 2 (similar to NPBWR2, formerly GPR8-like). Both NPB and NPW play a role in the control of feeding, neuroendocrine axis functions, memory and learning processes as well as in pain regulation. The present study aimed to investigate the expression of NPB, NPW, NPBWR1 and the similar to NPBWR2 genes in cultured rat calvarial osteoblast-like (ROB) cells and the effects of both peptides on proliferative activity and osteocalcin secretion by ROB cells. Classic RT-PCR technique revealed the presence of ppNPB mRNA, ppNPW mRNA, and NPBWR1 mRNA, but not similar to NPBWR2 mRNA in ROB cells. QPCR revealed gradual (days 7, 14 and 21 of culture) increase of the ppNPB gene expression, while expression of ppNPW gene was the highest at day 14 and was comparable to that seen in freshly isolated cells. In ROB cells, expression of NPBWR1 gene was notable at day 7 of culture, lower at day 21, and negligible at day 14. Neither NPB nor NPW changed osteocalcin secretion by cultured osteoblast-like cells while both neuropeptides inhibited their proliferative activity. Results of the present study suggest that the systems of NPW, NPB and NPBWR1 directly regulate proliferative activity of cultured rat calvaria osteoblast-like cells. The physiological significance of this osteoblastic system remains unclear, and requires further investigation.

Neuropeptide W has Cell Phenotype‐Specific Effects on the Excitability of Different Subpopulations of Paraventricular Nucleus Neurones

The administration of the neuropeptide W (NPW) and neuropeptide B (NPB) in rodents has been shown to influence the activity of a variety of autonomic and neuroendocrine systems. The paraventricular nucleus (PVN) is a major autonomic and neuroendocrine integration site in the hypothalamus, and neurones within this nucleus express the receptor for these ligands, NPB/W receptor 1 (NPBWR1). In the present study, we used whole cell patch clamp recordings coupled with single-cell reverse transcriptase-polymerase chain reaction to examine the effects of neuropeptide W-23 (NPW-23) on the excitability of identified PVN neurones. Oxytocin, vasopressin and thyrotrophin-releasing hormone neurones were all found to be responsive to 10 nm NPW-23, although both depolarising and hyperpolarising effects were observed in each of these cell groups. By contrast, corticotrophin-releasing hormone cells were unaffected. Further subdivision of chemically phenotyped cell groups into magnocellular, neuroendocrine or pre-autonomic neurones, using their electrophysiological fingerprints, revealed that neurones projecting to medullary and spinal targets were predominantly inhibited by NPW-23, whereas those that projected to median eminence or neural lobe showed almost equivalent numbers of depolarising and hyperpolarising cells. The demonstration of particular phenotypic populations of PVN neurones showing NPW-induced effects on excitability reinforces the importance of the NPB/NPW neuropeptide system as a regulator of autonomic function.

Neuropeptide W Influences the Excitability of Neurons in the Rat Hypothalamic Arcuate Nucleus

Neuropeptide W (NPW) is a ligand of the recently deorphaned receptor GPR7. Intracerebroventricular injection of this peptide results in reduced serum growth hormone concentration. Using whole-cell patch clamp recordings from somatostatin (SS) neurons in the hypothalamic arcuate nucleus, identified post-hoc using single-cell RT-PCR, we investigated the effects of NPW on membrane excitability. NPW application in acute slices of the arcuate nucleus resulted in the depolarization of the majority (62.5%) of the SS neurons tested, while smaller proportions of cells showed hyperpolarization or no response. Both the depolarization and hyperpolarization of arcuate SS neurons were preserved during recordings where voltage-gated sodium channels were blocked with tetrodotoxin, suggesting direct effects of NPW on the excitability of SS neurons. The observed depolarization of the majority of the SS neurons tested suggests that the central effects of NPW to inhibit growth hormone release results from activation of arcuate SS neurons, which could result in an inhibition of GHRH-releasing neurons.

Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach

Neuropeptide W (NPW), a novel endogenous peptide for G protein-coupled receptors GPR7 and GPR8, is expressed in the gastric antral mucosa of rat, mouse, and human stomachs. Here, we studied the ontogeny of NPW in the developing rat stomach. Real-time RT-PCR showed that NPW gene expression was initially detectable in embryonic day 14 (E14) stomach and gradually increased during the progress of age until birth, postnatal day 1 (P1). NPW mRNA level in the stomach increased again from the weaning period (P21) until reaching adulthood. Immunohistochemistry using polyclonal antibodies raised against rat NPW revealed that NPW-positive cells were detected in the P1 antral stomach and gradually increased during the development of age. Furthermore, double immunohistochemistry demonstrated that NPW colocalized with gastrin in P1 rat stomach. These data will provide clues to physiological functions of NPW in the development of rat stomach.

Expression of neuropeptide W in rat stomach mucosa: Regulation by nutritional status, glucocorticoids and thyroid hormones

Neuropeptide W (NPW) is a recently identified neuropeptide that binds to G-protein-coupled receptor 7 (GPR7) and 8 (GPR8). In rodent brain, NPW mRNA is confined to specific nuclei in hypothalamus, midbrain and brainstem. Expression of NPW mRNA has also been confirmed in peripheral organs such as stomach. Several reports suggested that brain NPW is implicated in the regulation of energy and hormonal homeostasis, namely the adrenal and thyroid axes; however the precise physiological role and regulation of peripheral NPW remains unclear. In this study, we examined the effects of nutritional status on the regulation of NPW in stomach mucosa. Our results show that in this tissue, NPW mRNA and protein expression is negatively regulated by fasting and food restriction, in all the models we studied: males, females and pregnant females. Next, we examined the effect of glucocorticoids and thyroid hormones on NPW mRNA expression in the stomach mucosa. Our data showed that NPW expression is decreased in this tissue after glucocorticoid treatment or hyperthyroidism. Conversely, hypothyroidism induces a marked increase in the expression of NPW in rat stomach. Overall, these data indicate that stomach NPW is regulated by nutritional and hormonal status.

Neuronal interactions between neuropeptide W- and orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexim- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.

Neuropeptide W in the rat pancreas: Potentiation of glucose-induced insulin release and Ca 2+ influx through L-type Ca 2+ channels in β-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10–100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1–100 nM) concentration-dependently increased cytosolic Ca 2+ concentration ([Ca 2+] i) at 8.3 mM glucose in rat single β-cells. The NPW-induced [Ca 2+] i increase was abolished under external Ca 2+-free conditions and by an L-type Ca 2+ channel blocker nifedipine (10 μM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing β-cells, but not glucagon-containing α-cells and somatostatin-containing δ-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates β-cells to enhance Ca 2+ influx through voltage-dependent L-type Ca 2+ channels and potentiates glucose-induced insulin release.

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus–pituitary–adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine- N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.

Neuropeptide W exerts a potent suppressive effect on blood leptin and insulin concentrations in the rat

Neuropeptide B/W receptor 1 (NPBWR1) and neuropeptide B/W receptor 2 (NPBWR2) are two structurally related orphan receptors linked to protein G. In rodents NPBWR2 is absent, and its counterpart is described as being similar to neuropeptide B/W receptor 2. Endogenous ligands of these receptors have been identified. One of them is 29 amino acid residues long, uniquely modified with bromine and, thus, termed neuropeptide B (NPB). The other, neuropeptide W (NPW), has been identified in two molecular forms of 23 and 30 amino acids (NPW23 and NPW30), respectively. Both NPB and NPW affect food intake and energy expenditure. Since leptin, a potent anti-obesity hormone, and insulin are involved in the control of energy homeostasis, the present study aimed to investigate whether NPB and NPW affect leptin and insulin secretion in the rat. RT-PCR technique revealed the presence of ppNPB, ppNPW, NPBWR1 and NPBWR2-like mRNAs in isolated pancreatic islets of the rat. NPB and NPW immunoreactivities were observed in all of the cells of the pancreatic islets. Only when a higher dose was administered (3 nmol/100 g body weight) did NPW transiently lower blood insulin levels whereas NPB injection did not alter insulinaemia in the studied rats. At 30 min, but not 60, of the experiment, NPW notably lowered blood leptin concentrations at both tested doses. On the contrary, NPB injections had no effect on blood leptin and insulin concentrations. Thus, the results suggest that NPW but not NPB exerts a potent suppressive effect on blood leptin concentrations in the rat, and this mechanism may be involved in NPW regulation of energy homeostasis.

Effects of intracerebroventricular administration of neuropeptide W30 on neurons in the hypothalamic paraventricular nucleus in the conscious rat

We demonstrated that intracerebroventricular (i.c.v.) administration of NPW30 increases the arterial blood pressure (ABP), heart rate (HR), and plasma catecholamine concentrations in conscious rats. NPW has been reported to be an important stress mediator in the central nervous system that modulates the hypothalamus–pituitary–adrenal (HPA) axis and sympathetic outflow. To examine the effects of NPW30 on the neural activity of the hypothalamic paraventricular nucleus (PVN), which is an integrative center of the autonomic and endocrine functions relevant to stress responses, we simultaneously recorded the single-unit activity in the PVN, ABP, and HR in conscious freely moving rats. Of the non-phasic (irregular) PVN neurons ( n = 35) examined, NPW30 (i.c.v. 3 nmol) elicited excitation in 22 neurons, inhibition in 7 neurons, and no response in 6 neurons, accompanied with increases in ABP and HR, whereas low-dose NPW30 (i.c.v. 0.3 nmol) did not affect the unit activity, ABP, or HR. Neurons that were affected by NPW30 were then further examined for their responses to perturbation in ABP and systemic administration of cholecystokinin-8 (CCK). The majority of neurons also showed responses to CCK, phenylephrine (PE), or nitroprusside (SNP). Our data suggest that central NPW30 modulates PVN neuronal activities, which might be involved in the regulation of cardiovascular function and energy balance through the autonomic nervous system, particularly, under stress-related conditions.

Neuropeptide W has bidirectional effects on the membrane potential of arcuate nucleus neurons

The previously orphaned receptor GPR7 and its endogenous ligand neuropeptide W (NPW) are found in the hypothalamic arcuate nucleus. Therefore, they have the potential to modulate a variety of autonomic processes via interaction with the neurons in this nucleus. Using a combination of whole cell patch clamp recording from hypothalamic brain slices containing the arcuate nucleus and single-cell reverse transcriptase polymerase chain reaction we have characterized the response of arcuate neurons to NPW and identified the phenotypes of responsive cells. Neurons expressing somatostatin, neuropeptide Y and POMC depolarized when exposed to nanomolar concentrations of NPW during current clamp recordings. This depolarization was often followed by a slowly developing membrane hyperpolarization. A minority of neurons hyperpolarized in the absence of depolarization. When arcuate neurons were pre-treated with the voltage-gated sodium channel blocker TTX, the depolarizing effect of NPW was largely inhibited while the slowly developing hyperpolarization was preserved. Consequently, NPW appears to have two main actions on neurons in the arcuate nucleus. Firstly, a rapidly induced release of excitatory neurotransmitter onto these neurons, followed, secondly, with a long-lasting hyperpolarization to ensure the depolarization is terminated with prolonged NPW exposure.

Immunofluorescent identification of neuropeptide B-containing nerve fibers and terminals in the rat hypothalamus

Neuropeptide B (NPB) and the structurally related neuropeptide W (NPW) have recently been identified as the endogenous ligands of the orphan G protein-coupled receptors GPR7 and GPR8. Whereas NPW is a high-affinity ligand for both GPR7 and GPR8, NPB activates only GPR7 in sub-nanomolar concentrations. GPR7 is highly conserved in both human and rodent orthologs while GPR8 has not been found in rodents. GPR7 mRNA is expressed in discrete regions of the hypothalamus suggesting a role in the regulation of energy homeostasis and neuroendocrine axes. In the present study, we have generated and extensively characterized antibodies that exert selective specificity for NPB. In dot-blot assays, these antibodies detected NPB but not NPW. Immunofluorescent staining of rat brain sections revealed moderately dense plexus of NPB-immunoreactive fibers and terminals in discrete areas of the hypothalamus. Neuronal somata were only seen in colchicine-treated rats. This immunostaining was completely abolished by preincubation of the antibodies with NPB but not with NPW. NPB-immunoreactivity was enriched in many regions within the hypothalamus which also contained high levels of GPR7 mRNA including the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, supraoptic retrochiasmatic nucleus, and in the area ventral to the zona incerta. Together, NPB and its receptor GPR7 exist in close proximity in the rat hypothalamus and are, hence, ideally positioned to modulate neuroendocrine functions.