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The central and peripheral neuropeptide Y (NPY) system is critically involved in feeding and energy homeostasis control. Disease conditions as well as aging can lead to reduced functionality of the NPY system and boosting it represents a promising option to improve health outcomes in these situations. Here we show that Ninjin-yoeito (NYT), a Japanese kampo medicine comprising twelve herbs, and known to be effective to treat anorexia and frailty, mediates part of its action via NPY/peptide YY (PYY) related pathways. Especially under negative energy homeostasis conditions NYT is able to promote feeding and reduces activity to conserve energy. These effects are in part mediated via signalling through the NPY system since lack of Y4 receptors or PYY leading to modification in these responses highlighting the possibility for combination treatment to improve aging related conditions on energy homeostasis control., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Perineuronal nets (PNNs) are extracellular matrix structures that form around some types of neurons at the end of critical periods, limiting neuronal plasticity. In the adult brain, PNNs play a crucial role in the regulation of learning and cognitive processes. Neuropeptide Y (NPY) is involved in the regulation of many physiological functions, including learning and memory abilities, via activation of Y1 receptors (Y1Rs). Here we demonstrated that the conditional depletion of the gene encoding the Y1R for NPY in adult forebrain excitatory neurons (Npy1r rfb mutant mice), induces a significant slowdown in spatial learning, which is associated with a robust intensification of PNN expression and an increase in the number of c-Fos expressing cells in the cornus ammonis 1 (CA1) of the dorsal hippocampus. Importantly, the enzymatic digestion of PNNs in CA1 normalizes c-Fos activity and completely rescues learning abilities of Npy1r rfb mice. These data highlight a previously unknown functional link between NPY-Y1R transmission and PNNs, which may play a role in the control of dorsal hippocampal excitability and related cognitive functions., (Copyright © 2020. Published by Elsevier Ltd.)

Background: The neuropeptide Y system affects various processes, among others food intake, and is frequently discussed in the context of targeting obesity. Studies in model organisms are indispensable to enable molecular studies in a physiological context. Although the NPY system is evolutionarily conserved in all bilaterians, in the widely used model Caenorhabditis elegans there is controversy on the existence of NPY orthologous molecules. While the FMRFamide-like peptide (FLP)/Neuropeptide receptor-Resemblance (NPR) system in the nematode was initially suggested to be orthologous to the mammalian NPY system, later global phylogenetic studies indicate that FLP/NPR is protostome-specific., Methods: We performed a comprehensive pharmacological study of the FLP/NPR system in transfected cells in vitro, and tested for functional substitution in C. elegans knockout strains. Further, we phenotypically compared different flp loss-of-function strains. Differences between groups were compared by ANOVA and post-hoc testing (Dunnett, Bonferroni)., Results: Our pharmacological analysis of the FLP/NPR system including formerly functionally uncharacterized NPY-like peptides from C. elegans demonstrates that G protein-coupling and ligand requirements for receptor activation are similar to the human NPY system. In vitro and in vivo analyses show cross-reactivity of NPY with the FLP/NPR system manifesting in the ability of the human GPCRs to functionally substitute FLP/NPR signaling in vivo. The high pharmacological/functional similarities enabled us to identify C. elegans FLP-14 as a key molecule in avoidance behavior., Conclusions: Our data demonstrate the pharmacological and functional similarities of human NPY and C. elegans NPR systems. This adds a novel perspective to current phylogenetic reconstructions of the neuropeptide Y system. NPY and NPR receptors are pharmacologically so similar that the human receptors can functionally compensate for the C. elegans ones, suggesting orthologous relationships. This is also underlined by the presence of NPY-like peptides and parallels in peptide requirements for receptor activation. Further, the results presented here highlight the potential of this knowledge for physiological as well as molecular studies on neuropeptide GPCRs such as the NPY system in the future.

Purpose: to explore whether the NK1 and Y2 receptors are involved in the pathogenesis of laser-induced CNV (choroidal neovascularization) in C57Bl/6N mice., Methods: CNV was induced by laser damage of Bruch's membrane and the CNV volume was determined by OCT and/or flatmount preparation. First, the development of the CNV volume over time was evaluated. Second, the CNV development in NK1- and Y2 KO mice was analyzed. Third, the effect on the development as well as the regression of CNV by intravitreal injections of the NK1 antagonist SR140333 and the Y2 antagonist BIIEO246 separately and each in combination with Eylea ® , was investigated. Furthermore, flatmount CNV volume measurements were correlated to volumes obtained by the in vivo OCT technique., Results: CNV volume peak was observed at day 4 after laser treatment. Compared to wild type mice, NK1 and Y2 KO mice showed significantly smaller CNV volumes. Eylea ® and the Y2 antagonist significantly reduced the volume of the developing CNV. In contrast to Eylea ® there was no effect of either antagonist on the regression of CNV, additionally no additive effect upon combined Eylea ® /antagonist treatment was observed. There was a strong positive correlation between CNV volumes obtained by OCT and flatmount., Conclusion: NK1 and Y2 receptors mediate the development of laser-induced CNVs in mice. They seem to play an important role at the developmental stage of CNVs, whereas VEGF via VEGF receptor may be an important mediator throughout the CNV existence. In vivo OCT correlates with flatmount CNV volume, representing a useful tool for in vivo evaluations of CNV over time., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Posttraumatic stress disorder is characterized by contextually inappropriate, dys-regulated and generalized fear expression and often resistant to therapy. The hippocampus integrates contextual information into spatial and emotional memories, but how diverse modulatory neurotransmitters are shaping this process is not known. Neuropeptide Y is a peptide-neurotransmitter, which modulates hippocampal excitability by activating several G-protein-coupled receptors. Postsynaptic Y1 receptors create strong anxiolytic and fear-suppressing behavior, while pre-synaptic Y2 receptors (Y2R) are mainly anxiogenic. The role of Y2Rs in spatial compared to emotional learning is, however, still controversial. Here we show that deletion of Y2Rs increased recall, but delayed extinction of contextual fear. Interestingly, spatial memory in the Barnes maze was enhanced during early and late testing, suggesting that Y2Rs suppress learning by hippocampal and extra-hippocampal mechanisms. To demonstrate sufficiency of hippocampal Y2Rs we performed viral vector-mediated, locally restricted re-expression of Y2Rs in the hippocampus of Y2KO mice. This treatment reduced spatial memory to the level of wildtype mice only during early, but not late recall. Furthermore, contextual fear was reduced, while induction of fear extinction appeared earlier. Our results suggest that hippocampal Y2R signaling inhibits learning in a time- and content-specific way, resulting in an early reduction of spatial memory and in a specific suppression of fear, by reducing fear recall and promoting fear extinction. We thus propose that reduction of hippocampal excitability through pre-synaptic Y2Rs may control the integration of contextual information into developing memories., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background and Purpose: Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut-brain peptides in modulating energy homeostasis and emotional-affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress-induced motor activity and anxiety by activating central Y4 receptors., Experimental Approach: We characterized [K(30) (PEG2)]hPP2-36 as long-acting Y4 receptor agonist and injected it peripherally into wildtype and Y4 receptor knockout (Y4KO) C57Bl/6NCrl mice to investigate the role of Y4 receptors in fear conditioning. Extinction and relapse after extinction was measured by spontaneous recovery and renewal., Key Results: The Y4KO mice showed impaired cued and context fear extinction without affecting acquisition, consolidation or recall of fear. Correspondingly, peripheral injection of [K(30) (PEG2)]hPP2-36 facilitated extinction learning upon fasting, an effect that was long-lasting and generalized. Furthermore, peripherally applied [K(30) (PEG2)]hPP2-36 before extinction inhibited the activation of orexin-expressing neurons in the lateral hypothalamus in WT, but not in Y4KO mice., Conclusions and Implications: Our findings suggests suppression of excessive arousal as a possible mechanism for the extinction-promoting effect of central Y4 receptors and provide strong evidence that fear extinction requires integration of vegetative stimuli with cortical and subcortical information, a process crucially depending on Y4 receptors. Importantly, in the lateral hypothalamus two peptide systems, PP and orexin, interact to generate an emotional response adapted to the current homeostatic state. Detailed investigations of feeding-relevant genes may thus deliver multiple intervention points for treating anxiety-related disorders., (© 2016 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The neuropeptide Y system is known to play an important role in the regulation of bone homeostasis and while the functions of its major receptors, Y1R and Y2R, in this process have become clearer, the contributions of other Y-receptors, like the y6 receptor (y6R), are unknown. Y6R expression is restricted to the suprachiasmatic nucleus (SCN) of the hypothalamus, an area known to regulate circadian rhythms, and the testis. Here we show that lack of y6R signalling, results in significant reduction in bone mass, but no changes in bone length. Male and female y6R knockout (KO) mice display reduced cortical and cancellous bone volume in axial and appendicular bones. Mechanistically, the reduction in cancellous bone is the result of an uncoupling of bone remodelling, leading to an increase in osteoclast surface and number, and a reduction in osteoblast number, osteoid surface, mineralizing surface and bone formation rate. y6R KO mice displayed increased numbers of osteoclast precursors and produced greater numbers of osteoclasts in RANKL-treated cultures. They also produced fewer CFU-ALP osteoblast precursors in the marrow and showed reduced mineralization in primary osteoblastic cultures, as well as reduced expression for the osteoblast lineage marker, alkaline phosphatase, in bone isolates. The almost exclusive location of y6Rs in the hypothalamus suggests a critical role of central neuronal pathways controlling this uncoupling of bone remodelling which is in line with known actions or other Y-receptors in the brain. In conclusion, y6R signalling is required for maintenance of bone mass, with loss of y6R uncoupling bone remodelling and resulting in a negative bone balance. This study expands the scope of hypothalamic regulation of bone, highlighting the importance for neural/endocrine coordination and their marked effect upon skeletal homeostasis., (Copyright © 2015. Published by Elsevier Inc.)

Y-receptors control energy homeostasis, but the role of Npy6 receptors (Npy6r) is largely unknown. Young Npy6r-deficient (Npy6r(-/-)) mice have reduced body weight, lean mass, and adiposity, while older and high-fat-fed Npy6r(-/-) mice have low lean mass with increased adiposity. Npy6r(-/-) mice showed reduced hypothalamic growth hormone releasing hormone (Ghrh) expression and serum insulin-like growth factor-1 (IGF-1) levels relative to WT. This is likely due to impaired vasoactive intestinal peptide (VIP) signaling in the suprachiasmatic nucleus (SCN), where we found Npy6r coexpressed in VIP neurons. Peripheral administration of pancreatic polypeptide (PP) increased Fos expression in the SCN, increased energy expenditure, and reduced food intake in WT, but not Npy6r(-/-), mice. Moreover, intraperitoneal (i.p.) PP injection increased hypothalamic Ghrh mRNA expression and serum IGF-1 levels in WT, but not Npy6r(-/-), mice, an effect blocked by intracerebroventricular (i.c.v.) Vasoactive Intestinal Peptide (VPAC) receptors antagonism. Thus, PP-initiated signaling through Npy6r in VIP neurons regulates the growth hormone axis and body composition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

This study was aimed to investigate the potential neuroprotective effect of neuropeptide Y (NPY) on the survival of dopaminergic cells in both in vitro and in animal models of Parkinson's disease (PD). NPY protected human SH-SY5Y dopaminergic neuroblastoma cells from 6-hydroxydopamine-induced toxicity. In rat and mice models of PD, striatal injection of NPY preserved the nigrostriatal dopamine pathway from degeneration as evidenced by quantification of (1) tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta, levels of (2) striatal tyrosine hydroxylase and dopamine transporter, (3) dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) as well as (4) rotational behavior. NPY had no neuroprotective effects in mice treated with Y(2) receptor antagonist or in transgenic mice deficient for Y(2) receptor suggesting that NPY effects are mediated through this receptor. Stimulation of Y(2) receptor by NPY triggered the activation of both the ERK1/2 and Akt pathways but did not modify levels of brain derived neurotrophic factor (BDNF) or glial cell line-derived neurotrophic factor. These results open new perspectives in neuroprotective therapies using NPY and suggest potential beneficial effects in PD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neuropeptide Y (NPY) has been extensively studied in relation to anxiety and depression but of the seven NPY receptors known to date, it is not yet clear which one is mainly involved in mediating its effects in emotional behavior. Mice lacking the NPY-Y2 receptors were previously shown to be less anxious due to their improved ability to cope with stressful situations. In the present study, the behavioral phenotype including the response to challenges was analyzed in NPY-Y2 knockout (KO) mice backcrossed in to congenic C57BL/6 background. In the elevated plus-maze (EPM) and the forced swim test (FST), the anxiolytic-like or antidepressant-like phenotype of the NPY-Y2 KO mice could not be confirmed, although this study differs from the previous one only with regard to the genetic background of the mice. In addition, no differences in response to acute stress or to the antidepressant desipramine in the FST were detected between wild type (WT) and NPY-Y2 KO animals. These results suggest that the genetic background of the animals appears to have a strong influence on the behavioral phenotype of NPY-Y2 KO mice. Additionally, to further characterize the animals by their biochemical response to a challenge, the neurochemical changes induced by the anxiogenic compound yohimbine were measured in the medial prefrontal cortex (mPFC) of NPY-Y2 KO and compared to WT mice. Dopamine (DA) levels were significantly increased by yohimbine in the WT but unaffected in the KO mice, suggesting that NPY-Y2 receptor exerts a direct control over both the tonic and phasic release of DA and that, although the anxiety-like behavior of these NPY-Y2 KO mice is unaltered, there are clear modifications of DA dynamics. However, yohimbine led to a significant increase in noradrenaline (NA) concentration and a slight reduction in serotonin concentration that were identical for both phenotypes., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Adult neurogenesis mainly occurs in two brain regions, the subventricular zone and the dentate gyrus (DG) of the hippocampus. Neuropeptide Y (NPY) is widely expressed throughout the brain and is known to enhance in vitro hippocampal cell proliferation. Mice lacking either NPY or the Y1 receptor display lower levels of cell proliferation, thereby suggesting a role for NPY in basal in vivo neurogenesis. Here, we investigated whether exogenous NPY stimulates DG progenitors proliferation in vivo. We show that intracerebroventricular administration of NPY increases DG cell proliferation and promotes neuronal differentiation in C57BL/6 adult mice. In these mice, the proliferative effect of NPY is mediated by the Y1 and not the Y2 receptor, as a Y1 ([Leu(31) ,Pro(34) ]), but not a Y2 (NPY(3-36) ), receptor agonist enhanced proliferation. In addition, no NPY-induced DG cellular proliferation is observed following NPY injection when coadministered with a Y1 antagonist or in the Y1 receptor knockout mouse. These results are in line with data obtained in Y1(-/-) mice, demonstrating that NPY regulates in vivo hippocampal neurogenesis. © 2010 Wiley-Liss, Inc., (Copyright © 2010 Wiley-Liss, Inc.)

Neuropeptide Y, Y1 receptors are found in neuronal as well as bone tissue and Y1 signalling has been implicated in the regulation of bone mass. However, the contribution of Y1 receptors located in these different tissues, particularly that of the bone-specific Y1 receptors, to the regulation of bone homeostasis is unclear. Here we demonstrate that osteoblast-specific Y1 receptor deletion resulted in a marked increase in femoral cancellous bone volume, trabecular thickness and trabecular number. This is the result of elevated osteoblast activity as shown by increased mineral apposition rate and bone formation rate, and is associated with an upregulation in the mRNA expression levels of alkaline phosphatase, osteocalcin and dentin matrix protein-1. Furthermore, osteoblastic Y1 receptor deletion also led to increased mineral apposition rate on both the endocortical and the periosteal surfaces resulting in increased femoral diameter. Together these data demonstrate a direct role for the Y1 receptor on osteoblasts in the regulation of osteoblast activity and bone formation in vivo and suggest that targeting Y1 receptor signalling directly in the bone may have potential therapeutic implications for stimulating bone accrual in diseases such as osteoporosis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The neuropeptide Y (NPY) system has been implicated in the regulation of bone homeostasis and osteoblast activity, but the mechanism behind this is unclear. Here we show that Y1 receptor signaling is directly involved in the differentiation of mesenchymal progenitor cells isolated from bone tissue, as well as the activity of mature osteoblasts. Importantly, the mRNA levels of two key osteogenic transcription factors, runx2 and osterix, as well as the adipogenic transcription factor PPAR-gamma, were increased in long bones of Y1(-/-) mice compared with wild-type mice. In vitro, bone marrow stromal cells (BMSCs) isolated from Y1(-/-) mice formed a greater number of mineralized nodules under osteogenic conditions and a greater number of adipocytes under adipogenic conditions than controls. In addition, both the number and size of fibroblast colony-forming units formed in vitro by purified osteoprogenitor cells were increased in the absence of the Y1 receptors, suggestive of enhanced proliferation and osteogenesis. Furthermore, the ability of two specific populations of mesenchymal progenitor cells isolated from bone tissue, an immature mesenchymal stem cell population and a more committed osteoprogenitor cell population, to differentiate into osteoblasts and adipocytes in vitro was enhanced in the absence of Y1 receptor signaling. Finally, Y1 receptor deletion also enhanced the mineral-producing ability of mature osteoblasts, as shown by increased in vitro mineralization by BMSCs isolated from osteoblast-specific Y1(-/-) mice. Together these data demonstrate that the NPY system, via the Y1 receptor, directly inhibits the differentiation of mesenchymal progenitor cells as well as the activity of mature osteoblasts, constituting a likely mechanism for the high-bone-mass phenotype evident in Y1(-/-) mice.

Expression levels of neuropeptide Y (NPY) are changed in schizophrenia patients. However, the direction of changes to NPY expression and the mechanisms behind NPY's impact on the development of the illness is not understood in detail. Here we investigated whether alterations in Y2 activity may be involved in the development of schizophrenia-related behaviours. We examined NPY Y2 receptor deficient male mice in behavioural domains relevant for the illness: locomotion, learning and memory, social interaction and sensorimotor gating (baseline and after acute challenge with psychotropic drugs) and the most relevant tasks were also completed in female Y2 mutants. Our investigations confirmed a hyper-locomotive phenotype for Y2 deficient male mice and no alterations in working and reference memory performance. Mutant males exhibited an increase in social interaction and moderately improved sensorimotor gating. The psychotropic drugs dexamphetamine and MK-801 affected prepulse inhibition similarly, whereas MK-801 appeared to be a slightly more potent stimulant for the acoustic startle response (ASR). Female Y2 deficient mice showed wild type-like performances in social interaction, working memory and prepulse inhibition. However, Y2 mutant females exhibited a moderately increased ASR compared to control mice. Taken together, lack of Y2 signalling in mice not only leads to altered locomotion but also changes social behaviours and affects sensorimotor gating. Thus, Y2 depletion influences a range of behaviours, which are potentially relevant for schizophrenia-related research., (Copyright 2009 Elsevier B.V. All rights reserved.)

Pancreatic polypeptide is released mainly from the pancreas, and is thought to be one of the major endogenous agonists of the neuropeptide Y Y(4) receptor. Pancreatic polypeptide has been shown to stimulate colonic muscle contraction, but whether pancreatic polypeptide has in vivo functional activity with respect to colonic transit is unclear. The present report investigated the effects of pancreatic polypeptide on fecal output as an index of colonic transit as well as intestinal motor activity, using wild-type (WT) and neuropeptide Y Y(4) receptor-deficient (KO) mice. Peripheral administration of pancreatic polypeptide increased fecal weight and caused diarrhea in WT mice in a dose-dependent manner (0.01-3mg/kg s.c.). Pancreatic polypeptide-induced increases in fecal weight and diarrhea completely disappeared in KO mice, while basal fecal weights did not differ between WT and KO mice. In longitudinal and circular muscles of mouse isolated colon, pancreatic polypeptide (0.01-1 microM) increased basal tone and frequency of spontaneous contraction in WT mice, but not in KO mice. Atropine did not affect pancreatic polypeptide-induced fecal output or increase in colonic muscle tone, indicating that the actions of pancreatic polypeptide are not mediated through cholinergic mechanisms. The present findings demonstrate that pancreatic polypeptide enhances colonic contractile activity and fecal output through neuropeptide Y Y(4) receptor, and a neuropeptide Y Y(4) receptor agonist might offer a novel therapeutic approach to ameliorate constipation., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Since no previous studies have reported dual measurements of stomach and duodenal motility in conscious mice, we developed a manometric method to measure the gastroduodenal motility in the physiological fed and fasted states of conscious mice. By this method we measured, for the first time, the gastroduodenal motility in Y2 knockout mice and analyzed the effects of ghrelin on the gastroduodenal motility in conscious mice. To evaluate this new method, we provide the comparison on the effects of CCK-8 examined by present and previous methods. In the fasted state of mice, phase III-like contractions with frequencies of 7.8 +/- 0.5 contractions/h in the antrum and 6.6 +/- 0.7 contractions/h in the duodenum were observed. This fasted pattern was disrupted and replaced by the fed pattern after feeding, with an increase of the motor index (MI) immediately after feeding. Intravenous injection of ghrelin induced the fasted pattern in the duodenum when injected in the fed state and increased %MI (114.3 +/- 9.8%) compared with saline-injected controls (64.4 +/- 9.6%) in the antrum. Intravenous injection of CCK-8 disrupted phase III-like contractions in both antrum and duodenum, which were replaced by fed-like motor patterns accompanied with the elevation of baseline pressure. In Y2 knockout mice, the frequency of phase III-like contractions was decreased in the antrum compared with wild-type mice and the immediate increase of MI after feeding seen in wild-type mice was disrupted in Y2 knockout mice. Our model provides a new method for studies of gastrointestinal motility in various mouse models, including transgenic and knockout ones.

In different behavioral paradigms including the elevated plus maze (EPM), it was observed previously that deletion of the neuropeptide Y Y2 receptor subtype results in potent suppression of anxiety-related and stress-related behaviors. To identify neurobiological correlates underlying this behavioral reactivtiy, expression of c-Fos, an established early marker of neuronal activation, was examined in Y2 receptor knockout (Y2(-/-)) vs. wildtype (WT) mice. Mice were placed on the open arm (OA) or closed arm (CA) of the EPM for 10 min and the effect on regional c-Fos expression in the brain was investigated. The number of c-Fos positive neurons was significantly increased in both WT and Y2(-/-) lines after OA and CA exposure in 51 of 54 regions quantified. These regions included various cortical, limbic, thalamic, hypothalamic, and hindbrain regions. Genotype influenced c-Fos responses to arm exposures in 6 of the 51 activated regions: the cingulate cortex, barrel field of the primary somatosensory cortex, nucleus accumbens, dorsal lateral septum, amygdala and lateral periaqueductal gray. These differences in neuronal activity responses to the novel environments were more pronounced after OA than after CA exposure. Mice lacking Y2 receptors exhibited reduced neuronal activation when compared to WT animals in response to the emotional stressors. Reduced neuronal excitability in the identified brain areas relevant to the processing of motivated, explorative as well as anxiety-related behaviors is suggested to contribute to the reduced anxiety-related behavior observed in Y2(-/-) mice., ((c) 2008 Wiley-Liss, Inc.)

There is growing evidence that neuropeptide Y (NPY) acting through Y1 and Y2 receptors has a prominent role in modulating anxiety- and depression-like behavior in rodents. However, a role of other Y-receptors like that of Y4 receptors in this process is poorly understood. We now investigated male Y2, Y4 single and Y2/Y4 double knockout mice in behavioral paradigms for changes in motor activity, anxiety and depression-like behavior. Motor activity was increased in Y2, Y4 and Y2/Y4 knockout mice under changing and stressful conditions, but not altered in a familiar environment. Y4 and Y2 knockout mice revealed an anxiolytic phenotype in the light/dark test, marble burying test and in stress-induced hyperthermia, and reduced depression-like behavior in the forced swim and tail suspension tests. In Y2/Y4 double knockout mice, the response in the light/dark test and in the forced swim test was further enhanced compared with Y4 and Y2 knockout mice, respectively. High levels of Y4 binding sites were observed in brain stem nuclei including nucleus of solitary tract and area postrema. Lower levels were found in the medial amygdala and hypothalamus. Peripheral administration of pancreatic polypeptide (PP) induced Y4 receptor-dependent c-Fos expression in brain stem, hypothalamus and amygdala. PP released peripherally from the pancreas in response to food intake, may act not only as a satiety signal but also modulate anxiety-related locomotion.

Important and novel roles for neuropeptide Y (NPY) signaling in the control of bone homeostasis have recently been identified, with deletion of either the Y1 or Y2 receptors resulting in a generalized increase in bone formation. Whereas the Y2 receptor-mediated anabolic response is mediated by a hypothalamic relay, the Y1-mediated response is likely mediated by osteoblastic Y1 receptors. The presence of Y1 receptors on osteoblasts and various other peripheral tissues suggests that, in addition to neuronal input, circulating factors may also interact with the Y1-mediated pathways. The skeletal and adipose tissue (peripheral and marrow) responses to Y1 receptor deficiency were examined after (1) leptin deficiency, (2) gonadectomy, and (3) hypothalamic NPY overexpression. Bone formation was consistently increased in intact Y1(-/-) mice. However, the hypogonadism of gonadectomy or leptin deficiency blocked this anabolism in male Y1(-/-) mice, whereas females remained unchanged. The Y1-mediated bone anabolic pathway thus seems to be dependent on the presence of intact androgen signaling. Y1 deficiency also led to increased body weight and/or adiposity in all experimental models, with the exception of male ob/ob, showing a general adipogenic effect of Y1 deficiency that is not dependent on androgens. Interestingly, marrow adipocytes were regulated differently than general adipose depots in these models. Taken together, this interaction represents a novel mechanism for the integration of endocrine and neural signals initiated in the hypothalamus and provides further insight into the coordination of bone and energy homeostasis.

Neuropeptide Y (NPY) is a potent neurotransmitter for feeding. Besides NPY, orexigenic neuropeptides such as agouti-related protein (AgRP), and anorexigenic neuropeptides such as alpha-melatonin stimulating hormone (MSH) and cocaine-amphetamine-regulated transcript (CART) are also involved in central feeding regulation. During fasting, NPY and AgRP gene expressions are up-regulated and POMC and CART gene expressions are down-regulated in hypothalamus. Based on the network of peptidergic neurons, the former are involved in positive feeding regulation, and the latter are involved in negative feeding, which exert these feeding-regulated peptides especially in paraventricular nucleus (PVN). To clarify the compensatory mechanism of knock-out of NPY system on feeding, change in gene expressions of appetite-related neuropeptides and the feeding behavior was studied in NPY Y5-KO mice. Food intake was increased in Y5-KO mice. Fasting increased the amounts of food and water intake in the KO mice more profoundly. These data indicated the compensatory phenomenon of feeding behavior in Y5-KO mice. RT-PCR and ISH suggested that the compensation of feeding is due to change in gene expressions of AgRP, CART and POMC in hypothalamus. Thus, these findings indicated that the compensatory mechanism involves change in POMC/CART gene expression in arcuate nucleus (ARC). The POMC/CART gene expression is important for central compensatory regulation in feeding behavior.

Loss of body weight is associated with bone loss, and body weight gain is associated with increased bone formation. The molecular mechanisms linking body weight, body composition, and bone density are now better understood. Lean mass is likely to have a significant, local effect on bone modeling and remodeling through mechanotransduction pathways. In contrast to the local regulation of bone formation and resorption by muscle-derived stimuli, peripheral body fat appears to influence bone mass via secretion of systemic, endocrine factors that link body weight to bone density even in non-weight bearing regions (e.g., the forearm). The cytokine-like hormone leptin, which is secreted by fat cells, is an important candidate molecule linking changes in body composition with bone formation and bone resorption. Increases in body fat increase leptin levels and stimulate periosteal bone formation through its direct anabolic effects on osteoblasts, and through central (CNS) effects including the stimulation of the GH-IGF-1 axis and suppression of neuropeptide Y, a powerful inhibitor of bone formation. Stimulation of beta2-adrenergic receptors through central (hypothalamic) leptin receptors does, however, increase remodeling of trabecular bone, resulting in a lower cancellous bone volume that may be better adapted to a concomitantly larger cortical bone compartment. These findings suggest that body weight and body fat can regulate bone mass and structure through molecular pathways that are independent of load-bearing. Furthermore, pharmacological manipulation of the signaling pathways activated by leptin may have significant potential for the treatment and prevention of bone loss.

Neuropeptide Y (NPY) is involved in the regulation of emotional behavior, and there is indirect evidence for a role of NPY in the cerebral responses to peripheral immune challenge. Since the NPY receptors involved in these reactions are not known, we investigated the effect of Escherichia coli lipopolysaccharide (LPS) on emotional, locomotor and social behavior, body temperature and circulating corticosterone in female Y2 (Y2-/-) and Y4 (Y4-/-) receptor knockout mice. LPS (0.1mg/kg injected IP 2.5h before testing) increased rectal temperature in control and Y4-/- mice to a larger degree than in Y2-/- animals. Both Y2-/- and Y4-/- mice exhibited reduced anxiety-related and depression-like behavior in the open field, elevated plus-maze and tail suspension test, respectively. While depression-like behavior was not changed by LPS, anxiety-related behavior was enhanced by LPS in Y2-/-, but not control and Y4-/- animals. Y2-/- mice were also particularly susceptible to the effect of LPS to attenuate locomotor behavior and social interaction with another mouse. The corticosterone response to LPS was blunted in Y2-/- mice which presented elevated levels of circulating corticosterone following vehicle treatment. These data show that Y2-/- mice are particularly sensitive to the effects of LPS-evoked immune stress to attenuate locomotion and social interaction and to increase anxiety-like behavior, while the LPS-induced rise of temperature and circulating corticosterone is suppressed by Y2 receptor knockout. Our observations attest to an important role of endogenous NPY acting via Y2 receptors in the cerebral response to peripheral immune challenge.

Recent evidence has shown that mice deficient in the NPY Y2 receptor have an increase in trabecular bone volume as well as cortical bone mass due to increased osteoblast activity. However, the mechanical phenotype of Y2 -/- bone has not yet been assessed. Thus, the aim of the present study was to examine the effect of Y2 deletion on murine cortical bone structural behavior, as well as to assess the material and geometric contributions to that behavior. The results of this study indicate that Y2 -/- mice on a 129 SV x Balb/c background strain are smaller in body mass and have smaller bones than wild-type controls. As expected based on smaller bone cross-sectional properties, cortical bone structural strength was lower in -/- animals. Surprisingly, the structural stiffness of -/- bones was comparable to that of +/+ bones despite their smaller cross-sectional geometry. Comparable structural stiffness appeared to be achieved by means of an elevated effective shear modulus, which was associated with a small, but statistically significant, higher ash content in Y2 -/- bones. These data represent the first comprehensive characterization of the effect of Y2 deletion on cortical bone structural and material behavior to date.

Unlabelled: NeuropeptideY-, Y2 receptor (Y2)-, and leptin-deficient mice show similar anabolic action in cancellous bone but have not been assessed in cortical bone. Cortical bone mass is elevated in Y2(-/-) mice through greater osteoblast activity. In contrast, leptin deficiency results in reduced bone mass. We show opposing central regulation of cortical bone., Introduction: Treatment of osteoporosis is confounded by a lack of agents capable of stimulating the formation of bone by osteoblasts. Recently, the brain has been identified as a potent anabolic regulator of bone formation. Hypothalamic leptin or Y2 receptor signaling are known to regulate osteoblast activity in cancellous bone. However, assessment of these pathways in the structural cortical bone is critical to understanding their role in skeletal health and their potential clinical relevance to osteoporosis and its treatment., Materials and Methods: Long bones of 16-week male ob/ob and germline and hypothalamic Y2(-/-) mice were assessed by QCT. Cortical osteoblast activity was assessed histologically., Results: The femora of skeletally mature Y2(-/-) mice and of leptin-deficient ob/ob and Y2(-/-)ob/ob mice were assessed for changes in cortical osteoblast activity and bone mass. Ablation of Y2 receptors increased osteoblast activity on both endosteal and periosteal surfaces, independent of leptin, resulting in increased cortical bone mass and density in Y2(-/-) mice along the entire femur. Importantly, these changes were evident after deletion of hypothalamic Y2 receptors in adult mice, with a 5-fold elevation in periosteal bone formation. This is in marked contrast to leptin-deficient models that displayed reduced cortical mass and density. These changes were associated with substantial differences in calculated strength between the Y2(-/-) and leptin-deficient mice., Conclusions: These results indicate that the Y2-mediated anabolic pathway stimulates cortical and cancellous bone formation, whereas the leptin-mediated pathway has opposing effects in cortical and cancellous bone, diminishing the production of cortical bone. The findings from conditional hypothalamic Y2 knockout show a novel, inducible control mechanism for cortical bone formation and a potential new pathway for anabolic treatment of osteoporosis.

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

The contribution of neuropeptide Y (NPY), deriving from adrenal medulla, to the adrenosympathetic tone is unknown. We found that in response to NPY, primary cultures of mouse adrenal chromaffin cells secreted catecholamine, and that this effect was abolished in cultures from NPY Y(1) receptor knockout mice (Y(1)-/-). Compared with wild-type mice (Y(1)+/+), the adrenal content and constitutive release of catecholamine were increased in chromaffin cells from Y(1)-/- mice. In resting animals, catecholamine plasma concentrations were higher in Y(1)-/- mice. Comparing the adrenal glands of both genotypes, no differences were observed in the area of the medulla, cortex, and X zone. The high turnover of adrenal catecholamine in Y(1)-/- mice was explained by the enhancement of tyrosine hydroxylase (TH) activity, although no change in the affinity of the enzyme was observed. The molecular interaction between the Y(1) receptor and TH was demonstrated by the fact that NPY markedly inhibited the forskolin-induced luciferin activity in Y(1) receptor-expressing SK-N-MC cells transfected with a TH promoter sequence. We propose that NPY controls the release and synthesis of catecholamine from the adrenal medulla and consequently contributes to the sympathoadrenal tone.

Neuropeptide Y has been implicated in pain modulation and is substantially up-regulated in dorsal root ganglia after peripheral nerve injury. To identify the role of neuropeptide Y after axotomy, we investigated the behavioral and neurochemical phenotype of neuropeptide Y Y1 receptor knockout mice with focus on dorsal root ganglion neurons and spinal cord. Using a specific antibody Y1 receptor immunoreactivity was found in dorsal root ganglia and in dorsal horn neurons of wild-type, but not knockout mice. The Y1 receptor knockout mice exhibited a pronounced mechanical hypersensitivity. After sciatic nerve axotomy, the deletion of Y1 receptor protected knockout mice from the axotomy-induced loss of dorsal root ganglion neurons seen in wild-type mice. Lower levels of calcitonin gene-related peptide and substance P were identified by immunohistochemistry in dorsal root ganglia and dorsal horn of knockout mice, and the axotomy-induced down-regulation of both calcitonin gene-related peptide and substance P was accentuated in Y1 receptor knockout. However, the transcript levels for calcitonin gene-related peptide and substance P were significantly higher in knockout than in wild-type dorsal root ganglia ipsilateral to the axotomy, while more calcitonin gene-related peptide- and substance P-like immunoreactivity accumulated proximal and distal to a crush of the sciatic nerve. These results indicate that the deletion of the Y1 receptor causes increased release and compensatory increased synthesis of calcitonin gene-related peptide and substance P in dorsal root ganglion neurons. Together, these findings suggest that, after peripheral nerve injury, neuropeptide Y, via its Y1 receptor receptor, plays a key role in cell survival as well as in transport and synthesis of the excitatory dorsal horn messengers calcitonin gene-related peptide and substance P and thus may contribute to pain hypersensitivity.

Neuropeptide (NPY) Y2 receptors play an important role in some anxiety-related and stress-related behaviours in mice. Changes in the level of anxiety can affect some cognitive functions such as memory, attention and inhibitory response control. We investigated the effects of NPY Y2 receptor deletion (Y2(-/-)) in mice on visual attention and response control using the five-choice serial reaction time (5-CSRT) task in which accuracy of detection of a brief visual stimulus across five spatial locations may serve as a valid behavioural index of attentional functioning. Anticipatory and perseverative responses provide a measure of inhibitory response control. During training, the Y2(-/-) mice had lower accuracy (% correct), and made more anticipatory responses. At stimulus durations of 2 and 4s the Y2(-/-) were as accurate as the Y2(+/+) mice but still more impulsive than Y(+/+). At stimulus durations of 0.25 and 0.5s both groups performed worse but the Y2(-/-) mice made significantly fewer correct responses than the Y2(+/+) controls. The anxiolytic drug diazepam at 2mg/kg IP greatly increased the anticipatory responding of Y2(-/-) mice compared to Y2(+/+). The anxiogenic inverse benzodiazepine agonist, FG 7142, at 10mg/kg IP reduced the anticipatory responding of Y2(-/-) but not Y2(+/+) mice. These data suggest that NPY Y2 receptors make an important contribution to mechanisms controlling attentional functioning and "impulsivity". They also show that "impulsivity" of NPY Y2(-/-) mice may depend on their level of anxiety. These findings may help in understanding the pathophysiology of stress disorders and depression.

Neuropeptide-Y (NPY) is involved in the regulation of ingestive behaviour and energy homeostasis. Since deletion of the NPY Y2 and Y4 receptor gene increases and decreases food intake, respectively, we examined whether water intake during the light and dark phases is altered in Y2 and Y4 receptor knockout mice. The water consumption of mice staying in their home cages was measured by weighing the water bottles at the beginning and end of the light phase during 4 consecutive days. Control, Y2 and Y4 receptor knockout mice did not differ in their water intake during the light phase. However, during the dark phase Y2 and Y4 receptor knockout mice drank significantly more (46-63%, P<0.05) water than the control mice. The total daily water intake over 24 h was also enhanced. The enhanced water intake during the dark phase was not altered by the beta-adrenoceptor antagonist propranolol or the angiotensin AT1 receptor antagonist telmisartan (each injected intraperitoneally at 10 mg/kg). These data indicate that NPY acting via Y2 and Y4 receptors plays a distinctive role in the regulation of nocturnal water consumption. While beta-adrenoceptors and angiotensin AT1 receptors do not seem to be involved, water intake in Y2 and Y4 receptor knockout mice may be enhanced because presynaptic autoinhibition of NPY release and inhibition of orexin neurons in the central nervous system are prevented.

Neuropeptide Y (NPY) in the CNS plays an important regulatory role in anxiety-related responses as exogenous administration of NPY exerts an anxiolytic-like effect in rodents. This effect is believed to be mediated by the Y(1) receptor system as pharmacological modulation of this Y(1) receptor system results in an increase in anxiety. Here we present a comprehensive phenotyping strategy for characterizing Y(1) receptor knockout animals at different times of the circadian rhythm using several motor activity-, exploration-, and anxiety-related behavioural tasks including open field, elevated plus maze, light-dark, and hole board test. We show that Y(1) deficiency has an important effect on motor activity and explorative-like behaviours and that it results in marked alterations in anxiety-related behaviours. Importantly, the behavioural phenotype of the Y(1) receptor knockout mice is circadian rhythm-dependent and also influenced by stimuli such as restraint stress. In addition, we found evidence for increases in working memory. Taken together, these findings suggest an important role of Y(1) receptors in the regulation of motor activity, exploration, and anxiety-related behaviours. This role is also influenced by several factors such as circadian rhythm and stress exposure confirming the importance of a comprehensive strategy and of using genetic animal models in behavioural neuroscience.

Neuropeptide Y receptors are critical regulators of energy homeostasis, but the functional interactions and relative contributions of Y receptors and the environment in this process are unknown. We measured the effects of an ad libitum diet of normal or high-fat food on energy balance in mice with single, double, or triple deficiencies of Y1, Y2, or Y4 receptors. Whereas wild-type mice developed diet-induced obesity, Y2Y4 double knockouts did not. In contrast, Y1 knockout or Y1Y2 or Y1Y4 receptor double knockout mice developed an exacerbated diet-induced obesity syndrome. Remarkably, the antiobesity effect of Y2Y4 deficiency was stronger than the obesogenic effect of Y1 deficiency, since Y1Y2Y4 triple knockouts did not develop obesity on the high-fat diet. Resistance to diet-induced obesity in Y2Y4 knockouts was associated with reduced food intake and improved glucose tolerance in the absence of changes in total physical activity. Fecal concentration of free fatty acids was significantly increased in Y2Y4 knockouts in association with a significantly reduced bile acid pool and marked alterations in intestinal morphology. In addition, hypothalamic proopiomelanocortin expression was decreased in diet-induced obesity (in both wild-type and Y1 receptor knockout mice) but not in obesity-resistant Y2Y4 receptor knockout mice fed a high-fat diet. Therefore, deletion of Y2 and Y4 receptors synergistically protects against diet-induced obesity, at least partially via changes in food intake and hypothalamic proopiomelanocortin expression.

The localization of the neuropeptide tyrosine (NPY) Y2 receptor (Y2R) protein was studied in mouse dorsal root ganglia (DRGs) and spinal cord, by using a recently developed rabbit anti-Y2R antibody and a sensitive immunohistochemical method. Y2R-like immunoreactivity (-LI) was observed in about 10% of the small/medium-sized lumbar DRG neurons. Among these, about 44% were calcitonin gene-related peptide-immunoreactive, and about 38% bound isolectin B4. In the dorsal horn of the spinal cord, an intense Y2R-LI was seen in the most superficial layers, mostly restricted to laminae I-II. This immunoreactivity was completely abolished by dorsal rhizotomy. Y2R-L1 was also detected on the skin, more abundantly in hairy than glabrous skin. Specificity experiments showed complete disappearance of the Y2R-LI described above after incubation with antibody preadsorbed with the immunogenic peptide. Furthermore, Y2R-LI was also absent in a Y2R knockout mouse. These results demonstrate that the NPY Y2R is associated mainly with both peptidergic and nonpeptidergic small, presumably nociceptive, neurons projecting to the superficial layers of the dorsal horn. The results also support a role for this receptor and NPY in pain mechanisms., ((c) 2005 Wiley-Liss, Inc.)

Adult dentate neurogenesis is important for certain types of hippocampal-dependent learning and also appears to be important for the maintenance of normal mood and the behavioural effects of antidepressants. Neuropeptide Y (NPY), a peptide neurotransmitter released by interneurons in the dentate gyrus, has important effects on mood, anxiety-related behaviour and learning and memory. We report that adult NPY receptor knock-out mice have significantly reduced cell proliferation and significantly fewer immature doublecortin-positive neurons in the dentate gyrus. We also show that the neuroproliferative effect of NPY is dentate specific, is Y1-receptor mediated and involves extracellular signal-regulated kinase (ERK)1/2 activation. NPY did not exhibit any effect on cell survival in vitro but constitutive loss of the Y1 receptor in vivo resulted in greater survival of newly generated neurons and an unchanged total number of dentate granule cells. These results show that NPY stimulates neuronal precursor proliferation in the dentate gyrus and suggest that NPY-releasing interneurons may modulate dentate neurogenesis.

The two neuropeptide Y (NPY) receptors Y1 and Y5 stimulate feeding in mammals, but are missing in the euteleosts, zebrafish and pufferfish (Takifugu rubripes). Both species have five other subtypes called Y2, Y7, Ya, Yb, and Yc. RT-PCR studies in pufferfish show that all five are expressed in the brain and may mediate NPY effects on feeding. Y2, Ya, and Yb are also broadly expressed in peripheral organs. These results reveal interesting differences in the NPY system of teleosts and mammals that may have arisen in the genetic turmoil involving the basal ray-fin fish tetraploidization.

Neuropeptide Y (NPY) is primarily synthesised and released by neurones, it is co-localised with noradrenaline and is involved in the regulation of cardiovascular function. In a mouse model lacking NPY Y1 receptor (KO), the ability of NPY to potentiate noradrenaline-induced vasoconstriction is abolished during stress but normal in baseline conditions, locomotor activity and metabolic rate are lowered, blood insulin levels and glucose storage activity are increased. The present study was aimed at further characterising NPY Y1 mutants, with special emphasis on: behavioural responses to novelty seeking and open-field with objects tests, heart rate responsiveness during acute social defeat, alpha2-adrenoceptor (alpha2-ARs) function in brain areas involved in cardiovascular regulation, and cardiac structure. As compared to wild-type controls (n=9), NPY Y1 KOs (n=9) showed: reduced somatomotor activation during non-social challenges, lower heart rate in baseline conditions, larger heart rate responsiveness during social defeat, increased number of alpha2-ARs in the dorsal motor nucleus of the vagus (nX) and the locus coeruleus (LC), moderately larger volume fraction of myocardial fibrosis. The remarkable increment of alpha2-adrenoceptor density in the nX and LC allows to view KO mice behavioural and anatomo-physiological peripheral characteristics as 'adaptations' to central adrenergic rearrangement due to NPY Y1 receptor deletion.

Mammalian circadian rhythms are modulated by neuropeptide Y (NPY), a peptide contained in the projection from the intergeniculate leaflet to the suprachiasmatic nuclei of the circadian pacemaker. NPY resets the circadian clock during the subjective day, mediating non-photic inputs. Previous studies using receptor-selective agonists have indicated that this action of NPY is mediated by the Y2 receptor in hamsters. The present study determined if NPY applied to the suprachiasmatic nuclei in the mid-subjective day can phase-advance the rhythm of spontaneous firing rate of Y2-/- mice. We observed that NPY did reset the rhythm of control mice but did not significantly shift the phase of this rhythm in the Y2-/- mice. These results provide strong evidence for the role of the Y2 receptor mediating neuropeptide Y subjective day phase-advance shifts in mice.

Vagal afferents signal gastric acid challenge to the nucleus tractus solitarii of the rat brainstem. This study investigated whether nucleus tractus solitarii neurons in the mouse also respond to gastric acid challenge and whether this chemonociceptive input is modified by neuropeptide Y acting via neuropeptide Y receptors of type Y2 or Y4. The gastric mucosa of female mice was exposed to different concentrations of HCl or saline, excitation of neurons in the nucleus tractus solitarii visualized by c-Fos immunohistochemistry, gastric emptying deduced from the gastric volume recovery, and gastric lesion formation evaluated by planimetry. Relative to saline, intragastric HCl (0.15-0.35 M) increased the number of c-Fos-expressing cells in the nucleus tractus solitarii in a concentration-dependent manner, inhibited gastric emptying but failed to cause significant hemorrhagic injury in the stomach. Mice in which the Y2 or Y4 receptor gene had been deleted responded to gastric acid challenge with a significantly higher expression of c-Fos in the nucleus tractus solitarii, the increases amounting to 39 and 31%, respectively. The HCl-induced inhibition of gastric emptying was not altered by deletion of the Y2 or Y4 receptor gene. BIIE0246 ((S)-N2-[[1-[2-[4-[(R,S)-5,11-dihydro-6(6H)-oxodibenz[b,e] azepin-11-yl]-1-piperazinyl]-2-oxoethyl]cyclopentyl] acetyl]-N-[2-[1,2-dihydro-3,5 (4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide; 0.03 mmol/kg s.c.), a Y2 receptor antagonist which does not cross the blood-brain barrier, did not modify the c-Fos response to gastric acid challenge. The Y2 receptor agonist peptide YY-(3-36) (0.1 mg/kg intraperitoneally) likewise failed to alter the gastric HCl-evoked expression of c-Fos in the nucleus tractus solitarii. BIIE0246, however, prevented the effect of peptide YY-(3-36) to inhibit gastric acid secretion as deduced from measurement of intragastric pH. The current data indicate that gastric challenge with acid concentrations that do not induce overt injury but inhibit gastric emptying is signaled to the mouse nucleus tractus solitarii. Endogenous neuropeptide Y acting via Y2 and Y4 receptors depresses the afferent input to the nucleus tractus solitarii by a presumably central site of action.

Y-receptor-knockout mice have become an important tool to elucidate specific physiological roles of individual Y-receptors. However, their phenotypes are not always confirmatory to results obtained by pharmacological investigations in vivo or in vitro. These discrepancies may, at least in part, be due to compensatory changes in the expression of remaining Y-receptor types. To determine whether deletion of individual Y-receptors results in altered mRNA expression and/or binding toward other Y-receptor types, we applied in-situ hybridization and radioligand-binding studies on brain slices of Npy1r-, Npy2r- or Npy5r-knockout mice. Significant changes were seen in Y1-receptor-deficient mice. Thus, Y2-receptor mRNA and (125)I-peptide YY(3-36) binding in the hippocampus proper were increased by up to 55% and 89%, respectively. Similar increases in (125)I-peptide YY(3-36) binding were observed in the caudo-dorsal extension of the lateral septum, an area heavily targeted by hippocampal projections and involved in Y1-receptor-regulated anxiety. Increased (125)I-peptide YY(3-36) binding and Y2-receptor mRNA levels were also observed in the medial amygdaloid nucleus. In contrast, (125)I-peptide YY(3-36) binding was reduced in the central amygdaloid nucleus. Y2-receptor mRNA in the intermediate part of the lateral septum was reduced by 42%. Only minimal changes were observed in Y2- or Y5-receptor-deficient mice. Our results demonstrate that compensatory changes in the expression of Y2-receptors occur in Y1-receptor-deficient mice. These adaptations are likely to contribute to changed physiological function. Thus, alterations in Y2-receptors have to be taken in account upon discussion of Y1-receptor function, especially in emotional aspects like anxiety and aggression, but also alcoholism.

Neuropeptide Y (NPY) blocks the effect of light on the mammalian circadian clock during the subjective night. The present study explores the role of the NPY Y1 receptor in this interaction. The effect of NPY when co-applied with NMDA, a glutamate agonist that can mimic the effect of light, was examined in NPY Y1-/- mice (background strain 129SVXBalb/c) using electrophysiology. Cells in the suprachiasmatic nucleus (SCN), the master circadian pacemaker, show a circadian rhythm in spontaneous firing rate that can be recorded in vitro. The results demonstrated that NPY attenuated the phase shifts to NMDA in both the Y1-/- mice and control mice, indicating that the Y1 receptor does not mediate the NPY blockade of photic-like phase shifts. The peak in frequency in the untreated control brain slices from Y1-/- mice was advanced by approximately 1 h as compared to the Y1+/+ mice. The Y1 receptor may contribute to a functional model of circadian rhythms, but apparently is not essential for the effects of NPY on photic phase shifts.

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation of a broad range of homeostatic functions. In the present work we studied NPY Y2 and Y5 receptor (R) gene expression in the mouse hypothalamus by using immunohistochemical detection of beta-galactosidase (beta-gal), a gene reporter molecule for Y2R and Y5R in Y2R-knockout (KO) and Y5R-KO mice, respectively. With this approach, cells normally expressing Y2R or Y5R are immunopositive for beta-gal. In the hypothalamus of the Y2R-KO mouse, beta-gal immunoreactivity (-ir) was found in numerous neurons of the medial preoptic nucleus as well as in the lateral anterior, periventricular, dorsomedial, tuberal, perifornical, and arcuate nuclei. Most of the dopaminergic neurons in the A13 dorsal hypothalamic group were beta-gal positive, whereas other hypothalamic dopaminergic neurons rarely displayed beta-gal-ir. In the arcuate nucleus, most of the beta-gal-positive neurons expressed NPY, but colocalizations with beta-endorphin were also found; in the tuberal and perifornical nuclei, many beta-gal-positive neurons contained nitric oxide synthase. beta-Gal-ir was also found in other forebrain regions of the Y2R-KO mouse, including the amygdala, thalamic nuclei, hippocampal CA3 area, and cortex. In the hypothalamus of the Y5R-KO mouse, beta-gal-positive neurons were found mainly in the arcuate nucleus and contained beta-endorphin. The present data show that Y2R and Y5R are expressed in distinct groups of hypothalamic neurons. High levels of Y2R expression in the preoptic nuclei suggest an involvement of Y2R in the regulation of reproductive behavior, whereas Y2R expression in the arcuate, dorsomedial, and perifornical nuclei may be relevant to feeding and body weight control. The finding that A13 dopaminergic neurons express Y2R suggests a new mechanism putatively involved in the central control of feeding, in which NPY can modulate dopamine secretion. The distribution of Y5R expression supports earlier evidence for involvement of this receptor in control of feeding and body weight via NPY's action on proopiomelanocortin-expressing neurons. J. Comp. Neurol. 470:256-265, 2004., (Copyright 2004 Wiley-Liss, Inc.)

Neuropeptide Y (NPY) and, in particular, the Y2 receptor subtype, has been suggested to be involved in learning and memory processing. However, the precise role of Y2 receptors in learning and memory remains unclear. In the present study, mice lacking NPY Y2-type receptors were assessed in two animal models of learning and memory processing. We found that NPY Y2-/- mice displayed a deficit on the probe trial in the Morris water maze task, whereas acquisition performance, swim speed, and visible platform performance did not differ significantly between groups. In addition, NPY Y2-/- mice exhibited a marked deterioration in object memory 6 h, but not 1 h, following initial exposure in the object recognition test. Both groups of mice showed similar locomotor activity profiles in a low-stress, open field test. These data support the hypothesis that Y2 receptors are involved in the regulation of learning and memory processing.

A complex system has evolved to regulate food intake and to maintain energy homeostasis. A series of short-term hormonal and neural signals that derive from the gastrointestinal tract, such as cholecystokinin (CCK), pancreatic polypeptide (PP) and peptide YY-(3-36), recently discovered to regulate meal size. Others such as ghrelin initiate meals, and insulin and leptin, together with circulating nutrients, indicate long-term energy stores. All these signals act on central nervous system sites which converge on the hypothalamus, an area that contains a large number of peptide and other neurotransmitters that influence food intake with neuropeptide Y (NPY) being one of the most prominent ones. Five Y receptors are known which mediate the action of neuropeptide Y and its two other family members, peptide YY and pancreatic polypeptide. Elevated neuropeptide Y expression in the hypothalamus leads to the development of obesity and its related phenotypes, Type II diabetes and cardiovascular disease. The limited availability of specific pharmacological tools and the considerable number of Y receptors have made it difficult to delineate their individual contributions to the regulation of energy homeostasis. However, recent studies analysing transgenic and knockout neuropeptide Y and Y receptor mouse models have started to unravel some of the individual functions of these Y receptors potentially also helping to develop novel therapeutics for a variety of physiological disorders including obesity.

New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippocampal learning. Little, however, is known about the control of hippocampal neurogenesis. We used primary hippocampal cultures from early post-natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neuropeptide Y is proliferative for nestin-positive, sphere-forming hippocampal precursor cells and beta-tubulin-positive neuroblasts and that the neuroproliferative effect of neuropeptide Y is mediated via its Y1 receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin-positive cells and beta-tubulin-positive cells in culture and short pulse 5-bromo-2-deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precursor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y-releasing interneurones.

Previously we showed that neuropeptide Y (NPY), a sympathetic vasoconstrictor neurotransmitter, stimulates endothelial cell migration, proliferation, and differentiation in vitro. Here, we report on NPY's actions, receptors, and mediators in ischemic angiogenesis. In rats, hindlimb ischemia stimulates sympathetic NPY release (attenuated by lumbar sympathectomy) and upregulates NPY-Y2 (Y2) receptor and a peptidase forming Y2/Y5-selective agonist. Exogenous NPY at physiological concentrations also induces Y5 receptor, stimulates neovascularization, and restores ischemic muscle blood flow and performance. NPY-mediated ischemic angiogenesis is not prevented by a selective Y1 receptor antagonist but is reduced in Y2(-/-) mice. Nonischemic muscle vascularity is also lower in Y2(-/-) mice, whereas it is increased in NPY-overexpressing rats compared with their WT controls. Ex vivo, NPY-induced aortic sprouting is markedly reduced in Y2(-/-) aortas and spontaneous sprouting is severely impaired in NPY(-/-) mice. NPY-mediated aortic sprouting, but not cell migration/proliferation, is blocked by an antifetal liver kinase 1 antibody and abolished in mice null for eNOS. Thus, NPY mediates neurogenic ischemic angiogenesis at physiological concentrations by activating Y2/Y5 receptors and eNOS, in part due to release of VEGF. NPY's effectiveness in revascularization and restoring function of ischemic tissue suggests its therapeutic potential in ischemic conditions.

Neuropeptide Y (NPY), a 36-aa peptide, is widely distributed in the brain and peripheral tissues. Whereas physiological roles of NPY as a hormoneneurotransmitter have been well studied, little is known about its other peripheral functions. Here, we report that NPY acts as a potent angiogenic factor in vivo using the mouse corneal micropocket and the chick chorioallantoic membrane (CAM) assays. Unlike vascular endothelial growth factor (VEGF), microvessels induced by NPY had distinct vascular tree-like structures showing vasodilation. This angiogenic pattern was similar to that induced by fibroblast growth factor-2, and the angiogenic response was dose-dependent. In the developing chick embryo, NPY stimulated vascular sprouting from preexisting blood vessels. When [Leu(31)Pro(34)]NPY, a NPY-based analogue lacking high affinity for the NPY Y(2) receptor but capable of stimulating both Y(1) and Y(5) receptors, was used in the corneal model, no angiogenic response could be detected. In addition, NPY failed to induce angiogenesis in Y(2) receptor-null mice, suggesting that this NPY receptor subtype was mediating the angiogenic signal. In support of this finding, the Y(2) receptor, but not Y(1), Y(4), or Y(5) receptors, was found to be widely expressed in newly formed blood vessels. Further, a delay of skin wound healing with reduced neovascularization was found in Y(2) receptor-null mice. These data demonstrate that NPY may play an important role in the regulation of angiogenesis and angiogenesis-dependent tissue repair.

Using a sensitive immunohistochemical technique, the localization of neuropeptide Y (NPY) Y1-receptor (Y1R)-like immunoreactivity (LI) was studied in various peripheral tissues of rat. Wild-type (WT) and Y1R-knockout (KO) mice were also analyzed. Y1R-LI was found in small arteries and arterioles in many tissues, with particularly high levels in the thyroid and parathyroid glands. In the thyroid gland, Y1R-LI was seen in blood vessel walls lacking alpha-smooth muscle actin, i.e., perhaps in endothelial cells of capillaries. Larger arteries lacked detectable Y1R-LI. A distinct Y1R-immunoreactive (IR) reticulum was seen in the WT mouse spleen, but not in Y1R-KO mouse or rat. In the gastrointestinal tract, Y1R-positive neurons were observed in the myenteric plexus, and a few enteroendocrine cells were Y1R-IR. Some cells in islets of Langerhans in the pancreas were Y1R-positive, and double immunostaining showed coexistence with somatostatin in D-cells. In the urogenital tract, Y1R-LI was observed in the collecting tubule cells of the renal papillae and in some epithelial cells of the seminal vesicle. Some chromaffin cells of adrenal medulla were positive for Y1R. The problem of the specificity of the Y1R-LI is evaluated using adsorption tests as well as comparisons among rat, WT mouse, and mouse with deleted Y1R. Our findings support many earlier studies based on other methodologies, showing that Y1Rs on smooth muscle cells of blood vessels mediate NPY-induced vasoconstriction in various organs. In addition, Y1Rs in other cells in parenchymal tissues of several organs suggest nonvascular effects of NPY via the Y1R., (Copyright 2002 Wiley-Liss, Inc.)

Aim: The aim of this review is to critically assess available evidence that blockade of the actions of NPY at one of the five NPY receptor subtypes represents an attractive new drug discovery target for the development of an appetite suppressant drug., Results: Blockade of the central actions of NPY using anti-NPY antibodies, antisense oligodeoxynucleotides against NPY and NPY receptor antagonists results in a decrease in food intake in energy-deprived animals. These results appear to show that endogenous NPY plays a role in the control of appetite. The fact that NPY receptors exist as at least five different subtypes raises the possibility that the actions of endogenous NPY on food intake can be adequately dissociated from other effects of the peptide. Current drug discovery has produced a number of highly selective NPY receptor antagonists which have been used to establish the NPY Y(1) receptor subtype as the most critical in regulating short-term food intake. However, additional studies are now needed to more clearly define the relative contribution of NPY acting through the NPY Y2 and NPY Y5 receptors in the complex sequence of physiological and behavioral events that underlie the long-term control of appetite., Conclusions: Blockade of the NPY receptor may produce appetite-suppressing drugs. However, it is too early to state with certainty whether a single subtype selective drug used alone or a combination of NPY receptor selective antagonists used in combination will be necessary to adequately influence appetite regulation.

We have shown previously that voluntary ethanol consumption and resistance to ethanol-induced sedation are inversely related to neuropeptide Y (NPY) levels in NPY-knock-out (NPY(-/-)) and NPY-overexpressing mice. In the present report, we studied knock-out mice completely lacking the NPY Y1 receptor (Y1(-/-)) to further characterize the role of the NPY system in ethanol consumption and neurobiological responses to this drug. Here we report that male Y1(-/-) mice showed increased consumption of solutions containing 3, 6, and 10% (v/v) ethanol when compared with wild-type (Y1(+/+)) control mice. Female Y1(-/-) mice showed increased consumption of a 10% ethanol solution. In contrast, Y1(-/-) mice showed normal consumption of solutions containing either sucrose or quinine. Relative to Y1(+/+) mice, male Y1(-/-) mice were found to be less sensitive to the sedative effects of 3.5 and 4.0 gm/kg ethanol as measured by more rapid recovery from ethanol-induced sleep, although plasma ethanol levels did not differ significantly between the genotypes. Finally, male Y1(-/-) mice showed normal ethanol-induced ataxia on the rotarod test after administration of a 2.5 gm/kg dose. These data suggest that the NPY Y1 receptor regulates voluntary ethanol consumption and some of the intoxicating effects caused by administration of ethanol.