Association of neuropeptide Y Y1 receptors with glutamate-positive and NPY-positive neurons in rat hippocampal cultures

The hippocampus is particularly enriched with neuropeptide tyrosine (NPY) and NPY receptors including the Y1, Y2 and Y5 subtypes. We have previously reported on the enrichment of cultured rat hippocampal neurons in specific [125I][Leu31, Pro34]PYY/BIBP3226-sensitive (Y1) binding sites and Y1 receptor mRNAs [St-Pierre et al. (1998) Br. J. Pharmacol., 123, p183]. We have now identified which cell types express the Y1 receptor. The majority of Y1 receptors, visualized using either the radiolabeled probe [125I][Leu31,Pro34]PYY or two antibodies directed against distinct domains of the Y1 receptor, was expressed in neurons as revealed by neuron-specific enolase (NSE) immunostaining. One antibody was directed against the second extracelllular loop of the Y1 receptor (amino acids 185-203) whereas the second was directed against the intracellular C-terminal loop (amino acids 355-382). The labelling was evident over both perikarya and processes. Neurons labelled by the various Y1 receptor probes were mostly glutamate-positive as revealed by double immunostaining. Most interestingly, a number of NPY-positive cultured hippocampal neurons were also enriched with the Y1 receptor, suggesting that this subtype may act as an autoreceptor to regulate NPY release in the hippocampus. These results thus provide an anatomical basis for the modulation of glutamate and NPY release by the Y1 receptor in the hippocampus.     

Infusion of neuropeptide Y into CA3 region of hippocampus produces antidepressant-like effect via Y1 receptor

A couple of papers indicate that patients with depression show a decrease in serum neuropeptide Y (NPY). To study the role of NPY in depression, we examined the effects of infusion of NPY into the hippocampus of learned helplessness (LH) rats (an animal model of depression). Infusion of NPY into the cerebral ventricle of LH rats showed antidepressant-like effects. Infusion of NPY into the CA3 region, but not the dentate gyrus (DG), produced antidepressant-like effects in the LH paradigm. Infusion of NPY did not affect locomotor activity or aversive learning ability. Coadministration of BIBO3304 (a Y1 receptor antagonist) with NPY to the CA3 region blocked the antidepressant-like effects of NPY, whereas coadministration of NPY with BIIE0246 (a Y2 receptor antagonist) to the CA3 region failed to block antidepressant-like effects. Furthermore, infusions of [Leu(31) Pro(34)]PYY (a Y1 and Y5 receptor agonist) alone and BIIE0246 alone into the CA3 region produced the antidepressant-like effects in LH rats. These results suggest that infusion of NPY into the CA3 region of hippocampus of LH rats produces antidepressant-like activity through Y1 receptors and attenuating effects through Y2 receptors.     

Sub-population of cultured hippocampal astrocytes expresses neuropeptide Y Y(1) receptors

The expression and pharmacological characterization of neuropeptide Y (NPY) receptors of the Y(1) subtype on cultured hippocampal neurons was reported using radioreceptor assays and immunohistochemical approaches (St-Pierre et al., 1998). The present study aimed to establish the presence of NPY Y(1) receptors on cultured hippocampal astrocytes using similar strategies. Immunocytochemical experiments were carried out using three antisera directed against distinct domains (amino acids sequence 185-203, 198-213 and 355-382) of the Y(1) receptor. Double-labeling experiments and confocal microscopy with these Y(1) receptor antisera demonstrated their recognition of the same sub-population (20%) of GFAP-positive astrocytes in culture. The immunostaining seen with all three Y(1) receptor antisera took the form of large irregular clusters distributed throughout cell bodies and processes. Further experiments using radioactive ligands confirmed the presence of NPY receptors on cultured hippocampal astrocytes. Emulsion receptor autoradiography using a newly developed ligand, [(125)I]GR231118 in the presence of PYY, hPP or BIBP3226 (1 microM), pharmacologically established the Y(1) nature of these receptors. Specific [(125)I]GR231118 binding was competed by PYY and the selective Y(1) antagonist BIBP3226 but not by hPP (a Y(4)/Y(5) agonist). Similar autoradiographic labeling patterns were obtained using [(125)I][Leu(31).Pro(34)]PYY (a Y(1)/Y(4)/Y(5) agonist) whereas [(125)I]PYY(3-36) (a Y(2)/Y(5) agonist) failed to generate any specific signal. Hence, rat cultured hippocampal astrocytes express a typical Y(1) receptor without evidence for the presence of Y(2), Y(4) or Y(5) subtypes. These data suggest a preferential regulation by NPY, acting via the Y(1) receptors, of astrocytic function.     

Characterization of a new neuropeptide Y Y5 agonist radioligand: [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP

In order to optimally characterize a class of neuropeptide Y (NPY) receptors expressed in a tissue enriched with multiple subtypes (Y1, Y2, Y4 and Y5) and to establish its detailed distribution, it is critical to use highly selective and specific probes that possess very low non-specific binding. In that context, we recently reported on the development of [125I][hPP(1-17), Ala31, Aib32]NPY as Y5 receptor radioligand. However, the non-specific binding obtained with this radioligand was too high to allow for detailed receptor autoradiography studies [Br. J. Pharmacol. 139 (2003) 1360]. Iodinated [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may represent a better Y5 radioligand in that regard. Accordingly, [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was investigated in rat brain membrane homogenates and its specificity and selectivity established in rat Y1, Y2, Y4 and Y5 transfected HEK293 cells. No specific binding was detected in HEK293 cells transfected with the rat Y1, Y2 or Y4 receptors, while saturable binding was observed in cells transfected with the rat Y5 receptor cDNA and in rat brain membrane homogenates (KD of 0.5-0.7 nM). Competition binding experiments performed in rat brain membrane homogenates demonstrated that specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was competed with nanomolar affinities by Y5 agonists and antagonists such as [Leu31,Pro34]PYY, PYY(3-36), [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, [Ala31, Aib32]NPY, [hPP(1-17), Ala31, Aib32]NPY, CGP71683A and JCF109, but not by Y1 (BIBP3226 and BIBO3304), Y2 (BIIE0246) and Y4 (GR231118) ligands. Non-specific binding was also lower than that reported for [125I][hPP(1-17), Ala31, Aib32]NPY. Interestingly, detailed analysis of competition binding curves obtained with [Leu31, Pro34]PYY, hPP, PYY(3-36) and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP against specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP sites were best fitted to a two-site model. Additionally, receptor autoradiography studies revealed the presence of specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding sites in the lateral septum and area postrema while other brain regions contained much lower levels of specific binding. Taken together, these data suggest that [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP represents a useful tool to study the unique feature of the Y5 receptor subtype.     

Regionally-selective down-regulation of NPY receptor subtypes in the obese Zucker rat. Relationship to the Y5 ‘feeding’ receptor

Experiments were performed to examine whether there are regionally and subtype selective changes in the density of neuropeptide Y (NPY) receptors in the obese Zucker rat which has an increased synthesis and release of NPY confined to the hypothalamus. Competition binding assays were employed to examine the feasibility of using [¹²⁵I]peptide YY ([¹²⁵I]PYY) to measure neuropeptide Y (NPY) Y5 ‘feeding’ receptors in the hypothalamus, hippocampal and cerebral cortex following masking of Y1 and Y2 receptors. Y5 receptors could not be discriminated from the binding to Y1 and Y2 receptors in hypothalamic, hippocampal or cerebral cortex homogenates, possibly owing to the small population of Y5 receptors expressed in the brain and the lack of selective ligands for this receptor. Quantitative receptor autoradiography was used to examine for regional changes in NPY receptor subtypes in obese versus lean Zucker rats. The non-selective Y1, Y2, Y4 and Y5 receptor ligand [¹²⁵I]PYY and the more selective Y1, Y4 and Y5 ligand [¹²⁵I][Leu³¹,Pro³⁴]PYY were employed, in conjunction with masking compounds in an attempt to measure any regional changes in the recently cloned NPY Y5 ‘feeding’ receptor. Specific [¹²⁵I]PYY and [¹²⁵I][Leu³¹,Pro³⁴]PYY binding was significantly reduced in the hypothalamic dorsomedial and arcuate nuclei as well as in the dorsal and lateral (perifornical) areas of obese Zucker rats, as compared to lean rats. In addition there were significant reductions in binding to the thalamic reuniens and centromedial nucleus, and hippocampal dentate gyrus of obese rats as compared to lean rats. Masking [¹²⁵I]PYY binding to Y1 receptors using 1 μM BIBP3226 demonstrated that the reduced NPY receptor density was due to reductions in Y2 or Y5 receptor density. The binding which was sensitive to BIBP3226, i.e. Y1 receptor density, was not different between obese and lean rats. Attempts using [¹²⁵I]PYY and the relatively selective Y2 agonist, [13–36]NPY to mask Y2 receptors and reveal Y5 receptors failed to leave any specific binding suggesting that [13–36]NPY was not selective enough to separate binding to Y2 and Y5 receptors. However, using [¹²⁵I][Leu³¹,Pro³⁴]PYY, masking binding to Y1 receptors using 1 μM BIBP3226 and masking any binding to Y4 using 1 nM rat pancreatic polypeptide left a small amount of binding remaining in the thalamus and hypothalamus, presumably to Y5 receptors which was significantly reduced in obese versus lean rat brain. These data suggest that there is a selective down-regulation in Y5 ‘feeding’ receptors in the obese Zucker rat which is known to possess a hyperactive arcuate-paraventricular NPY system.     

PYY and NPY: control of gastric motility via action on Y1 and Y2 receptors in the DVC

The pancreatic polypeptide family of hormones and neurotransmitters including pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY) are emerging as potent central regulators of gastric function. There is, however, considerable debate concerning the mechanisms and even the direction of effects mediated by these peptides. Good evidence exists showing that PYY is the ‘enterogastrone’ released by the ileum after feeding which acts on vagal reflex control circuits to reduce gastric motility (i.e. the ‘ileal brake’). However, equally convincing evidence is available to suggest that PYY and its close structural relative NPY may act in the same region of the brain to increase gastric motility through vagal mechanisms. We hypothesize that the confounding observations are due to agonist effects on two different receptor types – Y1 and Y2, which are both present in the dorsal vagal complex (DVC) but may be accessed differentially by peripheral humoral (PYY) vs central (NPY) pathways. In our initial approach to this problem, we studied the effects of NPY, PYY, Y1 and Y2 agonists microinjected into the DVC on gastric motility in the stimulated (by central thyrotropin-releasing hormone (TRH)) and basal conditions. Our results show that Y2 agonist applied to the DVC during conditions of TRH-stimulated gastric motility mimicked the suppressive effects of PYY applied under the same conditions. Under basal conditions, Y2 agonist has no effect on motility. The DVC effect of the Y1 agonist is the opposite; Y1 agonist has no further effect to stimulate gastric motility in the TRH stimulated condition while Y1 agonist strongly stimulates motility from the basal condition. The effects of NPY depend upon the condition of study. Under TRH stimulation (maximal motility), NPY in the DVC reduces (but does not completely suppress) gastric motility while in the basal state, NPY is a strong activator of motility. These results are discussed in terms of the possible differential localization of Y1 vs Y2 receptors within the DVC and in terms of recent findings suggesting that PYY is rapidly converted to a Y2 agonist by a ubiquitous dipeptidyl aminopeptidase (DAP-IV).     

Neuropeptide Y and Galanin Binding Sites in Rat and Monkev Lumbar Dorsal Root Ganalia and Spinal Cord and Effect of Peripheral Axotomy

Using monoiodinated peptide YY (PYY) and galanin as radioligands, and neuropeptide Y (NPY) fragments, the distribution of NPY binding sites and its subtypes Y1 and Y2, and of galanin binding sites, was investigated in rat and monkey lumbar (L) 4 and L5 dorsal root ganglia (DRG) and spinal cord before and after a unilateral sciatic nerve cut, ligation or crush. Receptor autoradiography revealed that [¹²⁵I]PYY bound to some DRG neurons and a few nerve fibres in normal rat DRG, and most of these neurons were small. NPY binding sites were observed in laminae I–IV and X of the rat dorsal horn and in the lateral spinal nucleus, with the highest density in laminae 1–11. [¹²⁵I]NPY binding was most strongly attenuated by NPY_13–36, a Y2 agonist, and partially inhibited by [Leu³¹,Pro³⁴]NPY, a Y1 agonist, in both rat DRG and the dorsal horn of the spinal cord. These findings suggest that Y2 receptors are the main NPY receptors in rat DRG and dorsal horn, but also that Y1 receptors exist. After sciatic nerve cut, PYY binding markedly increased in nerve fibres and neurons in DRG, especially in large neuron profiles, and in laminae III-IV of the dorsal horn, as well as in nerve fibres in dorsal roots and the sciatic nerve. Incubation with NPY_13–36 completely abolished PYY binding, which was also reduced by [Leu,³¹ Pro³⁴] NPY. However, the increase in PYY binding seen in laminae I–IV of the ipsilateral dorsal horn after axotomy was not observed after coincubation with [Leu³¹, Pro³⁴] NPY. NPY binding sites were seen in a few neurons in monkey DRG and in laminae I-II, X and IX of the monkey spinal cord. The intensity of PYY binding in laminae I-II of the dorsal horn was decreased after axotomy. Galanin receptor binding sites were not observed in rat DRG, but were observed in the superficial dorsal horn of the spinal cord, mainly in laminae I-II. Axotomy had no effect on galanin binding in rat DRG and dorsal horn. However, galanin receptor binding was observed in many neurons in monkey L4 and L5 DRG and in laminae I–IV and X of monkey L4 and L5 spinal cord, with the highest intensity in laminae I-II. No marked effect of axotomy was observed on the distribution and intensity of galanin binding in monkey DRG or spinal cord. The present results indicate that after axotomy the synthesis of NPY receptors is increased in rat DRG neurons, especially in large neurons, and is transported to the laminae I–IV of the ipsilateral dorsal horn and into the sciatic nerve. No such up-regulation of the NPY receptor occurred in monkey DRG after axotomy. The Y2 receptor seems to be the main NPY receptor in DRG and the dorsal horn of the rat and monkey spinal cord, but Y1 receptors also exist. The increase in NPY binding sites in laminae I–IV of the dorsal horn after axotomy partly represents Y1 receptors. In contrast to the rat, galanin binding sites could be identified in monkey lumbar DRG. No effect of axotomy on the distribution of galanin binding sites in rat or monkey DRG and dorsal horn was detected, suggesting their presence on local dorsal horn neurons (or central afferents).     

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T^3.40 was selected based on sequence alignments both between subtypes and between species and G^2.68, L^4.60 and Q^6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T^3.40I and Q^6.55A. Increased affinity was also observed for PYY to Q^6.55A. PYY(3-36) displayed decreased affinity for G^2.68N and L^4.60A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T^3.40I, L^4.60A and Q^6.55A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.     

Differential roles for neuropeptide Y Y1 and Y5 receptors in anxiety and sedation

Central administration of neuropeptide Y (NPY) causes both anxiolysis and sedation. Previous studies suggest that both effects are mediated via NPY Y1 receptors. However, most of these studies were carried out before the advent of specific NPY receptor ligands. Therefore, a potential role for other NPY receptors in anxiety and sedation remains a possibility. In the present study, we addressed this issue by testing the effects of intracerebroventricular (i.c.v.) injection of NPY as well as specific receptor agonists for the Y1 receptor ([D-His(26)]NPY), Y2 receptor (C2-NPY), and Y5 receptor ([cPP(1-7),NPY(19-23),Ala(31),Aib(32),Gln(34)]hPP) in the elevated plus maze and open field tests. As with NPY, the Y1 agonist had a dose-dependent anxiolytic-like effect in both behavioral tests. In contrast to NPY, which caused significant sedation in the open field test, the Y1 agonist was without sedative effect. The Y2 agonist showed neither anxiolytic-like nor sedative effects. The Y5 agonist showed anxiolytic-like activity in both behavioral tests and caused sedation in the same dose range as NPY in the open field test. These results indicate that anxiolytic-like effects of i.c.v.-administered NPY in rats are mediated via both Y1 and Y5 receptors, whereas sedation is mediated via Y5 receptors.     

Characterization of neuropeptide Y (NPY) receptors in human hippocampus.

We identified a 50 kDa neuropeptide Y (NPY) receptor from human hippocampus by affinity labeling. NPY specific binding and labeling of the receptor were inhibited in parallel by increasing concentrations of unlabeled NPY (IC50 = 0.27 nM and 0.18 nM, respectively). Peptide YY (PYY), but none of the pancreatic polypeptides, was as effective as NPY in displacing [125I]NPY. NPY fragments inhibited binding with the rank order of potency: NPY greater than NPY13-36 greater than NPY20-36 greater than or equal to NPY18-36 greater than NPY1-36 free acid greater than or equal to NPY26-36. These results demonstrate that the human hippocampal NPY receptor is a 50 kDa protein fitting the classification of a Y2 receptor subtype.     

The neurocircuitry and receptor subtypes mediating anxiolytic-like effects of neuropeptide Y

This review aims to give a brief overview of NPY receptor distribution and physiology in the brain and summarizes series of studies, test by test and region by region, aimed at identification receptor subtypes and neuronal circuitry mediating anxiolytic-like effects of NPY. We conclude that from four known NPY receptor subtypes in the rat (Y(1), Y(2), Y(4), Y(5)), only the NPY Y(1) receptor can be linked to anxiety-regulation with certainty in the forebrain, and that NPY Y(2) receptor may have a role in the pons. Microinjection studies with NPY and NPY receptor antagonists support the hypothesis that the amygdala, the dorsal periaqueductal gray matter, dorsocaudal lateral septum and locus coeruleus form a neuroanatomical substrate that mediates anxiolytic-like effects of NPY. The release of NPY in these areas is likely phasic, as NPY receptor antagonists are silent on their own. However, constant NPY-ergic tone seems to exist in the dorsal periaqueductal gray, the only brain region where NPY Y(1) receptor antagonists had anxiogenic-like effects. We conclude that endogenous NPY has an important role in reducing anxiety and serves as a physiological stabilizer of neural activity in circuits involved in the regulation of arousal and anxiety.     

Interaction of 125I-neuropeptide Y with rat cardiac membranes

125I-Neuropeptide Y (NPY) bound specifically with high affinity to rat atrial and ventricular membranes. Scatchard analysis revealed the presence of single class of binding sites in both atrial and ventricular membranes. The apparent Kd and Bmax for atrial membranes were 0.63 nM and 70 fmol/mg protein, respectively; ventricular membranes had an apparent kd of 0.39 nM and a Bmax of 283 fmol/mg protein. NPY structural homologues peptide YY (PYY) and pancreatic polypeptide (PP) bound to the ventricular membranes NPY receptor, but with several fold lower potency compared to NPY. Binding of 125I-NPY to ventricular membranes was sensitive to guanosine triphosphate (GTP) suggesting that the NPY receptor is linked to adenylate cyclase system. The receptor characterized in this system may play a crucial role in mediating the cardiac effects of NPY.     

The Y1 receptor subtype mediates the cardiovascular changes evoked by NPY administered into the posterior hypothalamic nucleus of conscious rat

An earlier study showed that the neuropeptide Y (NPY) receptor antagonist PYX-2 blocks the enhancement of a carbachol (CCh)-evoked pressor response produced by prior NPY administration into the posterior hypothalamic nucleus (PHN). The Y receptor subtype that mediates this response, and an increase in mean arterial pressure (MAP) and heart rate, remained unknown due to the lack of selectivity of PYX-2 for the Y receptor subtypes. Thus, the present study was undertaken to elucidate the Y receptor subtype responsible for mediating the NPY-evoked cardiovascular responses from the PHN by determining the rank order of potency of several NPY-related peptides for increasing MAP, and by correlating the pressor response evoked by these peptides to reported K(i)'s and IC(50)'s for the Y(1), Y(2), Y(4) and Y(5) receptor subtypes. The pharmacological profile (PYY>or=NPY>or=[Leu(31),Pro(34)]NPY>or=NPY(13-36)>or=hPP) and correlations suggest that the Y(1) and/or Y(5) receptor subtypes mediate these cardiovascular changes. Administration of the relatively non-selective Y receptor antagonist PYX-2 or the selective Y(1) receptor antagonist BIBP 3226 into the PHN prior to NPY completely blocked the cardiovascular responses. BIBP 3226 also blocked the cardiovascular changes evoked by [Leu(31),Pro(34)]NPY, NPY(13-36) and human pancreatic polypeptide (hPP). In contrast, neither BIBP 3226 nor PYX-2 inhibited the cardiovascular changes induced by peptide YY (PYY) or CCh microinjection into the PHN. These results show that NPY and PYY act on different receptors to mediate their respective cardiovascular changes from the PHN with NPY stimulating the Y(1) receptor.     

Role of Neuropeptide Y in the Regulation of Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels in the Male Rat Arcuate Nucleus as Evaluated by in situ Hybridization

Neuroanatomical data have clearly demonstrated the existence of synaptic contacts between neuropeptide Y (NPY) endings and tuberoinfundibular dopaminergic (TIDA) neurons in the rat arcuate nucleus. In order to determine the influence of NPY in the biosynthesis of dopamine, we have studied the effects of NPY and some NPY analogs on tyrosine hydroxylase (TH) gene expression in TlDA neurons in the male rat. The following peptides: NPY, PYY, [Leu³¹, Pro³⁴]-NPY (a Y, receptor agonist) and NPY_13–36 (a Y₂ receptor agonist) were injected into the left lateral ventricle of adult male rats. All the animals were perfused with 4% paraformaldehyde 4 h after injection. Cryostat sections through the arcuate nucleus were processed for quantitative in situ hybridization. The intracerebroventricular injection of NPY, PYY and [Leu³¹, Pro³⁴]-NPY induced an increased of 43, 33 and 42%, respectively, in the number of grains overlying TH neurons. On the other hand, the Y₂ receptor agonist NPY_13–36 did not influence mRNA levels. These data then strongly suggest that NPY positively regulates the genetic expression of TH in rat TlDA neurons via the Y, NPY receptor subtype.     

Neuropeptide Y selectively inhibits slow synaptic potentials in rat dorsal raphe nucleus in vitro by a presynaptic action.

Neuropeptide Y (NPY) has been shown to modulate synaptic transmission in both peripheral and central tissues via both pre- and postsynaptic mechanisms. In this study, we examined the effect of NPY and its analog, peptide YY (PYY), on slow synaptic potentials in the dorsal raphe nucleus in vitro using intracellular recording and single-microelectrode voltage-clamp techniques. NPY and PYY inhibited both the slow 5-HT1A receptor-mediated IPSP and the alpha 1-adrenoceptor-mediated slow EPSP while not affecting the fast, amino acid-mediated synaptic responses. PYY also inhibited pharmacologically isolated slow synaptic responses. NPY/PYY appear to mediate the observed inhibitions via a presynaptic mechanism, as the postsynaptic conductances mediated by activation of 5-HT1A receptors or alpha 1-adrenoceptors were unaffected by the peptides. NPY/PYY act via a different mechanism than presynaptic 5-HT1B receptors. NPY/PYY probably act via presynaptic Y2 receptors, as the C-terminal fragment NPY 13-36 and the Y2-selective agonist C2-NPY are effective. Since NPY and its receptors are present in the dorsal raphe nucleus, this peptide may act as an endogenous modulator of the state of activity of neurons in this region and may thus have a role in the modulation of neuronal output from this nucleus.     

NPY and Y receptors: lessons from transgenic and knockout models

Neuropeptide Y (NPY) in the central nervous system is a major regulator of food consumption and energy homeostasis. It also regulates blood pressure, induces anxiolysis, enhances memory retention, affects circadian rhythms and modulates hormone release. Five Y receptors (Y1, Y2, Y4, Y5 and Y6) are known to mediate the action of NPY and its two other family members, peptide YY (PYY) and pancreatic polypeptide (PP). Increased NPY signaling due to elevated NPY expression in the hypothalamus leads to the development of obesity and its related phenotypes, Type II diabetes and cardiovascular disease. Dysregulation in NPY signaling also causes alterations in bone formation, alcohol consumption and seizure susceptibility. The large number of Y receptors has made it difficult to delineate their individual contributions to these physiological processes. However, recent studies analysing NPY and Y receptor overexpressing and knockout models have started to unravel some of the different functions of these Y receptors. Particularly, the use of conditional knockout models has made it possible to pinpoint a specific function to an individual Y receptor in a particular location.     

Unaltered neuropeptide Y (NPY)-stimulated [35S]GTPgammaS binding suggests a net increase in NPY signalling after repeated electroconvulsive seizures in mice

Although electroconvulsive seizures (ECS) are widely used as a treatment for severe depression, the working mechanism of ECS remains unclear. Repeated ECS causes anticonvulsant effects that have been proposed to underlie the therapeutic effect of ECS, and neuropeptide Y (NPY) is a potential candidate for mediating this anticonvulsant effect. Repeated ECS results in prominent increases in NPY synthesis. In contrast, NPY-sensitive receptor binding is decreased, so it is unclear whether ECS causes a net increase in NPY signalling. Agonist-stimulated [35S]GTPgammaS binding is a method for detecting functional activation of G-protein-coupled receptors. The present study in mice examined the effects of daily ECS for 14 days on NPY-stimulated [35S]GTPgammaS functional binding and compared this with gene expression of NPY and NPY receptors as well as [125I]peptide YY (PYY) binding in hippocampus of the same animals. Significant increases in NPY mRNA and concomitant reductions in NPY-sensitive binding were found in the dentate gyrus, hippocampal CA1, and neocortex of ECS treated mice, which is consistent with previous rat data. These changes remained significant 1 week after repeated ECS. Significant increases in NPY Y1, Y2, and Y5 mRNA were found in the dentate gyrus after ECS. Surprisingly, unaltered levels of functional NPY receptor binding accompanied the decreased NPY-sensitive binding. This suggests that mechanisms coupling NPY receptor stimulation to G-protein activation could be augmented after repeated ECS. Thus increased synthesis of NPY after repeated ECS should result in a net increase in NPY signalling in spite of reduced levels of NPY-sensitive binding.     

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.     

Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry

Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPY's diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral-caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double-label immunocytochemistry indicate that Y1R immunoreactivity (-ir) and Y5R-ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1- and Y5-immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R- and Y5R-ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R-ir and Y1R-ir were detected in cells and fibers of the parvo- and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1-5 and C1-3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries.     

The discriminative stimulus effects of neuropeptide Y

Neuropeptide Y (NPY), an endogenous peptide which strongly induces food intake, is demonstrated to have discriminative stimulus properties when administered intracerebroventricularly. Rats rapidly learned to press the appropriate lever during training. NPY discrimination was dose-dependent. NPY's discriminative stimulus properties were compared to those of two doses of Peptide YY (PYY) and 24 and 48 h of food deprivation, conditions which also increase feeding. Both doses of PYY generalized to NPY, supporting previous findings that PYY has effects similar to NPY. Although food deprivation increases feeding in a manner similar to NPY, food deprivation did not result in NPY-appropriate responding.