Physiology and gene regulation of the brain NPY Y1 receptor

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Frontiers in Neuroendocrinology 27 (2006) 308–339

Frontiers inNeuroendocrinology

Review

Physiology and gene regulation of the brain NPY Y1 receptor

Carola Eva a,c,*, Mariangela Serra d,e,f, Paolo Mele a, GianCarlo Panzica b,c,Alessandra Oberto a

a Sezione di Farmacologia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita di Torino, Italyb Sezione di Anatomia, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Universita di Torino, Italy

c Centro Rita Levi Montalcini, Universita di Torino, Italyd Dipartimento di Biologia Sperimentale, Sezione di Neuroscienze, Universita di Cagliari, Cagliari, Italy

e Centro di Eccellenza per la Neurobiologia della Dipendenza, Universita di Cagliari, Cagliari, Italyf C.N.R. Istituto di Neuroscienze, Unita di Neuropsicofarmacologia, Cagliari, Italy

Abstract

Neuropeptide Y (NPY) is one of the most prominent and abundant neuropeptides in the mammalian brain where it interacts with afamily of G-protein coupled receptors, including the Y1 receptor subtype (Y1R). NPY-Y1R signalling plays a prominent role in the reg-ulation of several behavioural and physiological functions including feeding behaviour and energy balance, sexual hormone secretion,stress response, emotional behaviour, neuronal excitability and ethanol drinking. Y1R expression is regulated by neuronal activityand peripheral hormones. The Y1R gene has been isolated from rodents and humans and it contains multiple regulatory elements thatmay participate in the regulation of its expression. Y1R expression in the hypothalamus is modulated by changes in energetic balanceinduced by a wide variety of conditions (fasting, pregnancy, hyperglycaemic challenge, hypophagia, diet induced obesity). Estrogensup-regulate responsiveness to NPY to stimulate preovulatory GnRH and gonadotropin surges by increasing Y1R gene expression bothin the hypothalamus and the pituitary. Y1R expression is modulated by different kinds of brain insults, such as stress and seizure activity,and alteration in its expression may contribute to antidepressant action. Chronic modulation of GABAA receptor function by benzodi-azepines or neuroactive steroids also affects Y1R expression in the amygdala, suggesting that a functional interaction between theGABAA receptor and Y1R mediated signalling may contribute to the regulation of emotional behaviour. In this paper, we review thestate of the art concerning Y1R function and gene expression, including our personal contribution to many of the subjects mentionedabove.� 2006 Elsevier Inc. All rights reserved.

Keywords: NPY; Y1 receptor; Gene expression; Feeding behaviour; Reproductive behaviour; Anxiety; Stress; Epilepsy; Ethanol; Depression; GABA;Neuroactive steroids

1. Introduction

Neuropeptide Y (NPY) is a 36 amino acid peptide whichwas first isolated from pig brain by Tatemoto et al. [373].NPY is highly conserved through many species and,together with the peptide YY (PYY) and the pancreaticpolypeptide (PP), it forms the family of pancreatic peptides[373]. Immunohistochemical and in situ hybridization stud-

0091-3022/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.yfrne.2006.07.002

* Corresponding author. Fax: +39 0112367718.E-mail address: [email protected] (C. Eva).

ies have shown that NPY is one of the most abundant andwidely distributed peptides in the central nervous system(CNS) of both rodents and humans. NPY is abundantlyexpressed in various brain regions of rodents, includingthe hypothalamus, amygdala (Amy), hippocampus, nucle-us of the solitary tract (NST), locus coeruleus, nucleusaccumbens and the cerebral cortex [3,68,81,90]. NPY par-ticipates in the control of several physiological functions,including feeding behaviour, water consumption, learningand memory, locomotion, body temperature regulation,sexual behaviour, emotional behaviour, neuronal excitabil-ity, cardiovascular homeostasis, hormone secretion and

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circadian rhythms [14,72,123,144,158,260,364,392,395]. Inaddition, it has been suggested that NPY plays a role inpsychiatric disorders including depression, eating disor-ders, anxiety and epilepsy [14,72,144,260,395].

NPY interacts with a family of G-protein coupled recep-tors that includes the Y1, Y2, Y4, Y5, and y6 subtypes [247].The Y1 receptor (Y1R), Y2 receptor (Y2R) and Y5 receptor(Y5R) bind preferentially to NPY and PYY, whereas theY4 receptor (Y4R) shows selectivity towards the PP. They6 receptor (y6R) protein is truncated in most mammalsand it is functional only in the mouse and rabbit[112,223,314,365,401].

Among all the NPY receptors, the Y1R subtype hasreceived significant attention based on its ability to stimu-late feeding behaviour [177,183,259,415], inhibit nocicep-tion [268,433], regulate hormone secretion [31,173] andmodulate emotional behaviour, stress response [49,133]and ethanol drinking [332,361,375,376].

Our group was the first to clone the Y1R subtype forNPY in rats [99,199], and subsequently it was isolated fromhumans [141] and mice [101]. Sequence analysis of mam-malian and non-mammalian Y1R demonstrated that it dis-plays a high sequence homology in the transmembraneregions and it is highly conserved throughout evolution[207]. The most common actions of this receptor subtypeare the inhibition of adenylate cyclase, via the pertussis tox-in sensitive GTP binding protein Gi/Go and mobilizationof Ca2+ from intracellular stores [141]. Moreover, theY1R stimulates the mitogen-activated protein kinase(MAPK) pathways by inducing the phosphorylation ofextracellularly regulated kinase (ERK), an effect that hasbeen shown to be dependent on PI-3-kinase [228,270].

The Y1R is mainly located postsynaptically, although itcan be found at presynaptic sites. Several studies investigat-ed the localization of the Y1R within the mammalian CNS.The distribution of the Y1R was first analyzed by receptorautoradiography using [125I][Leu31, Pro34]PYY (Y1/Y4/Y5R-agonist), in the presence of the Y1R-antagonistBIBP3226 to distinguish Y1R from Y4/Y5R binding [89].In these studies, medium-high levels of Y1R-binding siteswere detected in the cerebral cortex, in the hippocampus(with particular intensity in the dentate gyrus), in the mam-illary nucleus, in the geniculate nucleus and in the NST.Low levels of binding sites were found in the septum, cer-ebellum, and in the paraventricular (PVN) and dorsomedi-al (DMH) hypothalamic nuclei. More recent studies in therat and mouse, using specific cRNA probes and in situ

hybridization techniques [193], detected a larger amountof positive nuclei, in particular in the thalamus, in the lim-bic system (hippocampus, Amy and bed nucleus of the striaterminalis) and in the hypothalamus [the medial preopticarea, PVN, DMH, ventromedial (VMH) and arcuate(ARC) nuclei]. Similar results were also collected inthe rat and mouse using specific antibodies against theC-terminal [197] or the N-terminal [417] of the Y1R.

The physiological effects of the Y1R were first discov-ered by pharmacological studies using selective agonists

and antagonists showing high binding affinities for thisreceptor subtype. However, the large number of NPYreceptors and the availability of few selective agonistsand antagonists have made it difficult to delineate Y1Rfunctions.

Y1R agonists were first identified by binding assays onstable transfected cells showing that peptides modified atthe C-terminal ([Pro34]NPY and [Leu31Pro34]NPY)retained their full activity for the Y1R but loose their affin-ity for the Y2R [5,39,199]. Later, it was discovered thatthese peptides were less selective than previously thoughtsince they displayed a significant affinity also for the Y5Rand Y4R. Novel compounds selectively binding the Y1Rwere subsequently synthesized, including [D-Arg25]-NPY,[D-His26]-NPY and Des-AA11�18[Cys7,21, D-Lys9(Ac),D-His26, Pro34]-NPY and [Phe7, Pro34]-NPY, the lattershowing it specifically activates the Y1R [259,358].

The first Y1R selective antagonist to be discovered wasBIBP3226, showing a Ki in the nanomolar range. Howev-er, its low solubility and oral availability reduced its valuefor in vivo experiments [317]. Other antagonists of the Y1Rhave been described, including BIB03304 that was shownto be more soluble and less toxic in vivo [409],SR120819A that displays affinity also for the Y4R andy6R [211,343] and GW1229 (also named 1229U91 orGR231118) that exhibits agonist properties for the Y4R[111]. More recently J-115814, a novel potent and selectiveY1R antagonist, was shown to act centrally on brain recep-tors when administered peripherally [176,361].

Knockout animal models for the Y1R have providedvalid alternatives to pharmacological approaches, althougha comparison of pharmacological and germ-line knockoutapproaches has shown some conflicting results [222]. Sever-al groups have generated Y1R knockout mouse models inwhich the Y1R coding sequence has been replaced by a cas-sette containing a neomycin-resistance gene by homolo-gous recombination [177,203,268,294]. The analysis ofmutated mice lacking the Y1R, as regards several physio-logical functions in which the Y1R has been implicated,has shown that phenotypes either exhibit minimal changesor they are conflicting when compared to pharmacologicalstudies.

For instance, deletion of the Y1R gene abolishes theability of NPY to potentiate noradrenaline-induced vaso-constriction, but fails to affect blood pressure [294].

In addition the central administration of Y1R agonistselicits a potent anxiolytic effect whereas mice lacking theY1R gene display anxious-like behaviour but only in cer-tain animal models [178]. Moreover, the infusion into thecentral amygdala (CeA) of the Y1R antagonist BIBP3226decreases alcohol consumption [332] whereas Y1R deficientmice exhibit increased voluntary ethanol consumption,when compared with wild type mice [376].

Additionally, despite the potent orexigenic action ofNPY, mutated mice lacking the Y1R do not display anymajor abnormalities in their feeding behaviour or bodyweight. Fasting-induced refeeding was diminished in Y1R

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knockout mice, but they show only a slight decrease of bas-al or NPY-induced food intake. Paradoxically, these micedevelop late onset obesity, with a significant increase inbody fat mass, and hyperinsulinemia, particularly in thefemales [177,203,294]. Interestingly, the most effective phe-notype alterations were observed in Y1R knockout micecarrying a non-functional leptin gene. For instance, theY1R deletion significantly reverts the obese phenotypeand the impairment of the gonadotropic axis function ofob/ob mice including significantly reduced fat mass,improved metabolic parameters, luteinizing (LH) hormonelevels and the onset of puberty [301].

These discrepancies between pharmacological studiesand germ-line knockout approaches have been attributedto the redundancy of systems that control different func-tions, such as energy homeostasis, which could lead tocompensatory changes during development. Accordingly,Wittmann and co-authors [416] have shown an increasein Y2R mRNA expression in the hippocampus and theAmy of Y1R knockout mice, suggesting that these adapta-tions might contribute to the altered phenotype of mutatedmice. Moreover, conflicting results derived from knockoutand non-genetic approaches may also be the consequenceof the deletion of the Y1R in all tissues where it is normallyexpressed, producing a phenotype that is compiled by thesum of all lost functions.

2. The Y1R/LacZ transgenic mouse model

Changes in the expression of neuropeptide proteins ormRNA as well as in their receptors are triggered by a pro-found stimulation or inhibition of the firing activity of theneuropeptide-containing neurons and probably reflectlong-term changes in neuronal circuits. Many questionsregarding the functional contribution of the Y1R to a par-ticular physiological process have been provided by studiesshowing dynamic changes of this receptor subtype inresponse to different conditions, including stress and anxi-ety, convulsions or changes in feeding behaviour. Forinstance, electrical kindling and kainic acid-induced epilep-sy decrease the Y1R and Y1R mRNA in rat hippocampus[117,194]. In the ARC food deprivation decreases andhyperphagia increases Y1R gene expression [67,169].

The Y1R, as well as the other members of the family ofG protein-coupled receptors, is highly regulated withrespect to its gene expression, resulting in a temporallyand spatially specific pattern of distribution. To study in vi-

vo the regulation of Y1R gene expression at the transcrip-tional level we generated, as a model, the transgenicmouse line Y1R/LacZ carrying the 1.3 kb 5 0 flanking regionof the mouse Y1R promoter fused to the coding region ofthe Escherichia coli LacZ gene (Fig. 1H) [275]. Analysisof Y1R/LacZ transgene activity by histochemical stainingwith X-gal demonstrated b-galactosidase expression in spe-cific brain regions (Fig. 1A–G), while no LacZ expressionwas evidenced in white matter or in peripheral tissues. Fourtransgenic lines showed characteristic patterns of b-galac-

tosidase activity in the brain that are consistent with theexpression of the endogenous gene. Moreover, the ontoge-netic analysis indicated that the transgenic constructappears to be activated with the same timing of the endog-enous Y1R during the development of the embryonicnervous system. These data demonstrated that the 1.3 kbupstream region of the murine Y1R contains the cis actingelements required to replicate the expression pattern of theendogenous Y1R gene in a CNS-restricted and develop-mental stage-specific manner in vivo.

The use of b-galactosidase staining with chromogenicsubstrate X-gal in transgenic mouse lines not only facili-tates the study of the spatiotemporal expression of a neuro-peptide receptor gene, but it can be usefully employed toquantitatively evaluate in vivo changes in transgene expres-sion following, for instance, a neuronal lesion or a pharma-cological treatment. Modern computer-assistedmorphometrical techniques [318] may in fact be utilizedto automatically quantify the number of dye precipitateswithin a selected region, making a numerical measurementpossible which is proportional to the histochemical activity(based on the presence) of the expressed transgene. Thesecalculations are not equivalent to direct gene product anal-ysis (i.e. blot analysis, in situ hybridization), but areextremely useful for statistical comparison of differenttreatments. By using this approach, we demonstrated thatpharmacological treatments can modulate Y1R/LacZ

transgene expression in a tissue-specific manner, suggestingthat changes in the transgene expression may reflect chang-es of Y1R steady state and, therefore, they can be used as amarker of altered NPY-Y1R signal transduction [100].

In this review, we will describe changes in the regionaland functional expression pattern of the Y1R protein,mRNA and Y1R/LacZ transgene expression as an impor-tant step toward understanding the physiological role ofthis receptor subtype during development or in brainfunction.

3. The transcriptional regulation of the Y1R gene

Extensive studies have investigated the molecular mech-anisms involved in the regulation of Y1R expression andfunction at the transcriptional and posttranscriptionallevel. Endogenously expressed [246] or transfected[113,145] Y1R rapidly desensitizes in response to prolongedstimulation with agonists via activation of multiple effec-tors including PKC, tyrosine kinase, the 7TM receptorkinase GRK and the interaction with b2 arrestin[29,146,370]. Moreover the Y1R is internalized inside thecell when occupied by agonists through a pathway depen-dent on clatrin coated pits and subsequent sorting of thereceptor to recycling endosomes [113,293,296].

The Y1R gene was first isolated from a mouse genomiclibrary in our laboratory [101] and these studies weresubsequently extended by others in humans [140].

The Y1R gene is clustered together with the Y5R onmouse chromosome 8B3-C2 and on human chromosome

Fig. 1. (A–C) Drawings of mouse brain coronal sections arranged in a rostro-caudal extent illustrating the expression of Y1R/LacZ transgene (line 62).Dots represent the b galactosidase positive cells; their number is proportional to the intensity of labelling. Arc, arcuate nucleus; BST, bed nucleus of thestria terminalis; BSTS, bed nucleus of the stria terminalis, supracapsular division; CA1, CA2, CA3, different regions of the hippocampus; Cg, cingulatecortex; Cl, claustrum; Cpu, caudate putamen; DLG, dorsal lateral geniculate nucleus; DMH, dorsomedial hypothalamic nucleus; En, endopiriformnucleus; GrDG, granule layer of the dentate gyrus; MeP, medial amygdaloid nucleus, posterior part; MPOM, medial preoptic nucleus, medial part; Pir,piriform cortex; PLCo, posterolateral cortical amygdaloid nucleus; PMCo, posteromedial cortical amygdaloid nucleus; PVN, paraventricular nucleus;SFO, subfornical organ; SON, supraoptic nucleus; VMH, ventromedial hypothalamic nucleus. Redrawn and simplified from [275]. (D–G) b galactosidaseactivity in the medial amygdala and hypothalamus of transgenic mice (line 62) expressing Y1R/LacZ. (D–E) Dense labelling in the region of the bednucleus of the stria terminalis (BST), the medial preoptic nucleus (MPOM), and the paraventricular nucleus (PVN). (F) less intense labelling in theposterior hypothalamus [arcuate nucleus (Arc), ventromedial (VMH) and dorsomedial (DMH) hypothalamic nuclei]. (G) intense labelling of the posterioramygdaloid complex [medial (MeP) and posteromedial cortical (PMCo) nuclei]. (H) Schematic representation of the murine Y1R gene and the Y1R/LacZ

transgene used to generate transgenic mice. Upper part: Murine Y1R gene. Exons are indicated by solid boxes and numbers. Lower part: Schematicdiagram of the SalI BglII Y1R/LacZ transgene. Arrows indicate the start sites of transcription. Restriction enzyme sites in the 5 0 flanking region of themurine Y1R gene are indicated. Modified from [275].

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4q31 and they are transcribed in opposite directions from acommon promoter region suggesting that they haveevolved from a gene duplication event [141,264]. The tran-scription of both genes from opposite strands of the sameDNA sequence suggests that transcriptional activation ofone of the receptors could modulate the regulation of geneexpression of the other. As both Y1R and Y5R are thought

to play an important role in the regulation of food intake,coordinate expression of their specific genes may be impor-tant in the modulation of NPY activity.

The coding region of the gene is interrupted by twointrons in all species explored, one in the 5 0-untranslatedregion and an 80 bp internal intronic sequence [101,140]that has been shown to enhance the expression of both

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the Y1R and Y5R in vitro [230]. The expression of the Y1Rhas been suggested to be under the control of at least threedifferent promoters that are activated in a tissue-specificmanner [11]. The existence of multiple transcripts of thehuman Y1R mRNA, which are generated by the alternativesplicing of three different exons, was also demonstrated[264].

The regulatory region of the Y1R gene has been isolatedin mice, humans and rats [40,101,140] and regulation of thecell (or tissue) specific expression of this gene at the level oftranscription has been analyzed. Reporter gene assays per-formed in our laboratory demonstrated that a 1.3 kb frag-ment of the 5 0 flanking region of the murine Y1R gene isable to direct specific expression of the LacZ reporter genein neuron-derived cells [101].

Comparison of the 5 0 flanking sequence of cloned Y1Rgenes with the transcription factor database has shown thatit contains consensus sequences for transcription factorsaffected by cAMP and phorbol esters, glucocorticoidsand estrogens. Furthermore, potential binding sitesfor the transcription factor NF-kB were found, suggesting

Fig. 2. Upper panel: Luciferase fusion construct containing the 1.3 kb regulatopositions (relative to the ATG) of the proximal nucleotide in putative consencAMP (CRE), glucocorticoids (GRE), estrogens (ERE) and NF-jB (Y1-kappaLuciferase fusion construct containing mutations of both ERE1 and ERE2flanking region is shown below. CRE, cAMP responsive element; ERE, estropanel: (A) Effect of estrogen receptor (ER) agonist and antagonist on the transactivity was determined in NG108-15 cells transiently transfected with the p13plasmid together with the ERa expression vector and treated with vehicle (bestradiol + 10 nM ICI 182 780 (dark grey bars) for 24 h. (B) Mutational analyactivity driven by the Y1R gene promoter region. NG108 cells were co-transfecfusion constructs containing mutations of both ERE1 and ERE2 half palindrwith vehicle (black bars) or with 0.1 nM 17b-estradiol (light grey bars) for 24

that its expression is tightly regulated[11,40,101,140,262,263,275] (Fig. 2).

The Y1R promoter contains one, non-palindromic glu-cocorticoid responsive element (GRE) site and severalreverse complement non-palindromic GRE sites that areconserved in mouse, human and rat receptor genes[11,40,101]. Reporter gene assays have shown the transcrip-tional activity of the rat Y1R gene promoter is stimulatedby dexamethasone in PC12 cells suggesting that the imper-fect GRE sites on the Y1R gene may act co-operatively[40]. This finding is consistent with the observation thatglucocorticoids cause an up-regulation of Y1R expressionin the ARC of rats [2,208], although it is not known if thisis a direct effect of the glucocorticoids acting on the Y1Rgene promoter. Moreover, the administration of glucocor-ticoids increases the density of the Y1R in the adrenocorti-cal mouse cell line [405], Y-1 [404] and PC12 cells [83].

The cAMP–PKA signalling pathway stimulates thetranscriptional activity of the Y1R gene in PC12 and SK-N-BE2 neuroblastoma cell lines and this effect is complete-ly blocked by the treatment with an inhibitor of PKA

ry region of the murine Y1R gene (p1305-LUC). Nucleotide sequences andsus sequences for transcription factors affected by phorbol esters (AP1),B) residing in the 5 0 flanking region of the murine Y1R gene are indicated.half palindromic sites (pm1/m2ERE-LUC) of the murine Y1 receptor 5 0

gen responsive element; GRE, glucocorticoid responsive element. Lowercriptional activity of the Y1R gene promoter in NG108-15 cells. Luciferase05-LUC reporter plasmid (p1305-LUC), or with the p1305-LUC reporterlack bars), 0.1 nM 17b-estradiol (light green bars) or with 0.1 nM 17b-sis of ERE emipalindromic sites on 17b-estradiol stimulation of luciferaseted with the expression vector for the human ERa together with luciferaseomic sites (pm1/m2ERE-LUC). Transiently transfected cells were treatedh. *p < 0.05 versus vehicle (Newman–Keuls test). Modified from [263].

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[40,345]. Moreover, the stimulation of the Y1R increasescAMP responsive element binding protein (CREB) phos-phorylation and induces the expression of cAMP respon-sive element (CRE) containing genes, including the Y1Rgene, via mobilization of Ca2+ and activation of CaMKs[344,346]. Conversely the Y1R inhibits the expression ofCRE containing genes via the inhibition of the cAMP path-ways only when cAMP levels are elevated. Consideringthat decreased function of CREB in the CeA and in themedial amygdala (MeA) may regulate anxiety and alcoholabuse via decreased expression of NPY [284,289], the regu-lation of Y1R gene expression by phosphorylated CREmight also be physiologically relevant.

The transcription factor NF-jB was found to play arole in the regulation of the transcriptional activity ofthe murine Y1R gene. We demonstrated that the promot-er region of the Y1R gene contains consensus sites formembers of the NF-jB/Rel family of transcription factorsthat bind jB-related complexes in nuclear extracts fromrat brain areas and NG108-15 cells and behave as enhanc-er elements when placed upstream of deletion fragmentsof the Y1R regulatory region [262]. In most cell typesthe jB-related proteins mediate an immediate-earlyresponse to stimuli that represent stress conditions. NPYseems to play a critical role in the transmission ofstress-related informations to the hypothalamic/hypophy-sial system and in the activation of neuroendocrineresponses essential for the survival of the organism (seebelow). An interesting possibility is that the Y1R forNPY may represent one of the jB site-containing genesthat are modulated in the mammalian nervous systemby jB-related factors in response to stimuli that requirean immediate defensive response.

We also demonstrated that Y1R gene transcription intransiently transfected neuroblastoma glioma cells wasstimulated by approximately twofold in response to 17b-es-tradiol treatment (Fig. 2A) [263]. The enhancement wasdose-dependent, blocked by an estrogen receptor antago-nist, and strictly dependent on the presence of estrogenreceptor-a. Mutational analysis has shown that two ofthe half-palindromic estrogen responsive elements (EREs)flanking the Y1R gene are responsible for conferring estra-diol inducibility to the Y1R gene promoter (Fig. 2B). Thisregulatory mechanism may be physiologically relevantsince estrogens were found to increase the responsivenessto NPY of gonadotropin-releasing hormone (GnRH) neu-rons and of gonadotropes through the stimulation of Y1Rgene expression [143,425]. In addition, changes in Y1Rgene expression have been detected during the estrous cyclein female Y1R/LacZ transgenic mice [232].

4. Y1R and feeding behaviour

Feeding behaviour plays a crucial role in the regulationof energy homeostasis and it is tightly regulated by severalneuronal, metabolic and endocrine signals that are inte-grated by the hypothalamus.

Short-term hormonal and neural signals that derivefrom the gastrointestinal tract, including the cholecystoki-nin (CCK), pancreatic polypeptide (PP), peptide YY-(3–36) and grelin, control meal size. Long-term regulation ofenergy stores is provided by the main circulating hor-mones, insulin and leptin. All these signals act via path-ways converging on the hypothalamus, an area thatcontains a large number of anabolic and catabolic neuro-transmitters, as well as neuropeptides with NPY beingone of the most prominent ones.

NPY is the prototype hormone to stimulate feedingbehaviour, inducing, in particular, the intake of carbohy-drates [362,403]. Within the hypothalamus, NPY is mainlysynthesized in neurons whose cell bodies lie in the ARC orDMH nuclei and send projections to adjacent areas, suchas PVN, perifornical hypothalamus (PHF), VMH hypo-thalamic nuclei, and the lateral hypothalamic area (LH)that are involved in the daily regulation of ingesting behav-iour and energy balance [3,9,81] (Fig. 3B). There are alsoshort projections that terminate within the ARC, whereNPY modulates the activity of both NPY and anorexigenicproopiomelanocortin (POMC) neurons in an inhibitorymanner. In addition to the ARC neurons, NPY is also syn-thesized in the dorsovagal complex of the medulla, whichreceives afferent nerves from the gastrointestinal tract andproject to the PVN and to the DMH [3,9,81].

Early evidence demonstrated that NPY, after injectioninto either the cerebral ventricles or the hypothalamus,exerts a powerful stimulatory effect on food intake evenin satiated rats, eventually leading to obesity[69,217,362,364,389,403]. NPY also decreases thermogene-sis, in brown adipose tissue (BAT), through inhibition ofsympathetic outflow to BAT [34]. Subsequently, evidencefor a physiological role of NPY in the control of feedingbehaviour and body weight has been obtained from thedemonstration that a close relationship exists between foodintake and NPY expression levels in the hypothalamus. Inpoor metabolic conditions, such as starvation, lactation[356] or insulin deficient diabetes, hypothalamic NPY syn-thesis and release in the ARC/PVN neurons are increased[87,106,172,326,414]. Moreover, the blockade of NPYeffects by immuno-neutralization or NPY depletionsuppresses feeding and decreases obesity [88,97,340].

Leptin and insulin are synthesized peripherally and arereleased in the plasma in proportion to the extension ofthe body fat, and they act centrally to inhibit feeding andto increase energy expenditure [57,128]. Among the orexi-genic neuronal systems, NPY is a prime candidate implicat-ed in mediating leptin action in the hypothalamus. Distinctneuronal populations in the ARC express leptin (Ob)receptors and coexpress various neuropeptides includingNPY [85,334,366]. In vivo studies showed that leptin inhib-its the synthesis and release of NPY [335] and counteractsthe effect of NPY on feeding, whereas NPY opposes theanorectic effect of leptin. In rodent models of genetic obes-ity [ob/ob, db/db, Zucker (fa/fa), Koletsky (f/f) and corpu-lent (cp/cp) JCR LA], due to leptin deficiency, there is an

Fig. 3. Distribution of Y1R (A, blue stars) and NPY (B and C, black dots) within the rodent brain. Based on [193,197,275] for Y1R and [68] for NPY. (B)Blue arrows indicate the hypothalamic NPY pathway involved in feeding behaviour (based on [9,76]). (C) Brain NPY pathways thought to be involved inNPY-effects related to stress and emotionality (based on [134]). Abbreviations: Amy, amygdala; AON, anterior olfactory nucleus; Arc, arcuate nucleus;BST, bed nucleus of the stria terminalis; Cb, cerebellum; DB, diagonal band of Broca; DMH, dorsomedial hypothalamic nucleus; Hip, hippocampus; IC,inferior colliculus; LC, locus coeruleus; LH, lateral hypothalamic nucleus; MPOM, medial preoptic nucleus; NST, nucleus of the solitary tract; PAG,periaqueductal gray; PVN, paraventricular nucleus; SC, superior colliculus; VE, vestibular complex; VMH, ventromedial hypothalamic nucleus; VTA,ventral tegmental area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)

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over production of NPY in the hypothalamus that has beenshown to be partially responsible for the hyperphagia anddecreased energy expenditure seen in these mice[21,162,240,329,334,413]. In addition, the ablation of theNPY gene decreases hyperphagia and obesity in ob/ob

mice, and an increased sensitivity to the anorexigenic effectof leptin is observed in mice deficient for NPY expression[97,98].

Similarly, the expression of NPY in rat ARC neuronsdecreased after the central administration of insulin,whereas ARC NPY neurons become overactive when thelevels of this hormone fall during undernutrition[322,338,351]. Insulin replacement in streptozotocin diabet-ic rats normalizes the increase in hypothalamic NPYexpression and reduces hyperphagia that develops second-ary to insulinopenia [241,407]. It has been shown that, inthe presence of physiological increases in the plasma insulin

concentrations, stimulation of hypothalamic insulin signal-ling is sufficient for the inhibition of endogenous glucoseproduction. This effect may be mediated by the insulin-in-duced down regulation of hypothalamic NPY neuronalactivity [276,277,387].

Specific regions of the hypothalamus or of the brainstemcontain ‘glucose-sensing’ neurons that can detect changesin glucose availability [120,198,261]. Glucose-sensitiveNPY neurons were identified in medial ARC where bothNPY and AgRP are released, and NPY neurons are appar-ently sensitive to glucose and activated by hypoglycaemia[261,295,412]. Thus glucose may represent another negativefeedback signal beside leptin or insulin that regulates theactivity of the NPY neurons.

On the other hand, ARC NPY neurons also contain glu-cocorticoid receptors and NPY mRNA and NPY immuno-reactivity are affected by corticosterone in the PVN as well

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as in the ARC [17,374]. The enhanced corticosterone circu-lating levels that occur in starvation and diabetes increasehypothalamic NPY mRNA expression [410]. Moreover,adrenalectomy reduces hyperphagia and weight gain inobese Zucker rats and VMH-lesioned rats, and decreaseshyperphagia and insulin release elicited by NPY; in turn,corticosterone treatment of adrenalectomized rats increasesNPY gene expression and reverts these effects[2,82,208,363].

The brain–gut peptide CCK is released upon nutrientstimulation of neuroendocrine cells lining the intestinallumen [257]. Peripheral exogenous CCK reduces the sizeand duration of a meal by acting at the level of theNST, a brain area that receives primary vagal afferentinput [339]. Moreover, the DMH contains both CCK neu-rons and CCK-A receptors and has been suggested to be acentral site of CCK action [24]. Several lines of evidencesuggest a negative relationship between NPY and CCKin the regulation of feeding behaviour (for review see[189]). NPY reduces both CCK-induced satiety and itsability to increase c-Fos-like- immunoreactivity in NST,suggesting that NPY may, in part, increase food intakeby decreasing the response to NST neurons to input relat-ed to satiety [242,333]. On the other hand, neurons withinthe compact subregion of the DMH coexpress NPY andCCK-A receptors, and local CCK administration reducesfood intake and decreases the DMH NPY mRNA expres-sion [24]. Furthermore, studies performed on OtsukaLong–Evans Tokushima fatty (OLETF) rats, lackingCCK-A receptors, have shown an increased expressionof NPY in the DMH, suggesting that a deficit in CCK’saction in the control of the DMH NPY gene expressionmay play a major role in the obese and hyperphagic phe-notype of these rodents [32].

In addition to these circulating factors, NPY is coex-pressed and/or makes reciprocal connections with severalorexigenic and anorexigenic neurotransmitters. NPY andnoradrenaline and adrenaline are co-expressed in brainstem neurons projecting to the PVN [321,324]. NPY, AgrPand GABA are co-expressed in ARC neurons and they actsynergistically to enhance feeding [44,47,127]. In addition,NPY regulates in an inhibitory manner, through differentmechanisms, the anorexigenic melanocortin neurons onthe ARC/PVN axis (for review see [45]). Thus, in theARC, NPY exerts an inhibitory tone directly over theneighbouring melanocortin neurons. AGRP, co-releasedfrom NPY/AGRP containing neurons, can block theanorexigenic melanocortin signalling whereby the antago-nism of MC3 and MC4 receptors in the ARC and PVN,respectively. Similarly, it has been suggested that GABA,co-released from NPY/AGRP neurons, inhibits POMCneurons by acting in the ARC as well as in the PVNthrough different receptors [75,148,306].

Data summarized above strongly indicate that NPY is aphysiological appetite transducer and that it represents thehypothalamic target for most metabolic and hormonal sig-nals. Therefore, a large number of studies have been done

to determine the receptor subtypes involved in the orexi-genic effect of this peptide [158,160,291]. Of the six receptorcloned to date, the orexigenic NPY receptors are the Y1Rand Y5R subtypes [158]. Both of these receptors areexpressed in the hypothalamic sites involved in the dailyregulation of ingesting behaviour and energy balance, i.e.the ARC, PVN, DMH, VMH and LH [90,193] (Fig. 3A).The importance of the Y1R subtype in feeding behaviourwas first shown by pharmacological studies using selectiveagonists and antagonists. In rats, intracerebroventricular(ICV) administration of NPY peptide derivatives, that bindwith high affinity to Y1R, were shown to stimulate feeding[259] and to induce hyperinsulinemia independently onfood intake [108]. BIBP3226 and BIBO3304, two selectiveY1R antagonists, block appetite stimulation elicited byICV administration of NPY [183,409]. These observationshave been recently confirmed by the observation that theperipheral administration of a highly selective Y1R antag-onist inhibits NPY-induced feeding and that this com-pound is devoid of activity when administered to Y1Rknockout mice [176].

Demonstration that the NPY-Y1R signalling plays aprominent role in the stimulation of feeding and obesityalso comes from the observation that changes in feedingbehaviour and energy balance induce a marked plasticityin the Y1R function and expression in specific regions ofthe hypothalamus.

In this regard, we used Y1R/LacZ transgenic mice toinvestigate the effect of changes of energetic balance,induced by fasting and glucose administration, on theY1R gene expression at the transcriptional level [430](Table 1).

We demonstrated that fasting for 72 h, which increasesARC NPY mRNA, also induces a marked decrease inthe Y1R/LacZ transgene expression in the PVN. Leptin,that blunts the fasting-induced increase of NPY mRNA,prevents the down regulation of Y1R gene expressioninduced by food deprivation [430]. These observations sug-gest that the stimulation of NPY orexigenic signal, elicitedby a state of negative energetic balance such as starvation,is mediated by the Y1R in PVN and that the target cellsmediating the restraint by leptin on NPY-induced feedingresponse resides in this nucleus. Interestingly, leptin revers-es the fasting-induced decrease in Y1R expression in PVNbut fails to decrease mice body weight, suggesting thatthe inhibition of ARC NPY neurons and the weight reduc-tion are triggered by leptin at different sensitivities [1,430].The fasting-induced activation of the ARC NPY systemwas also found to decrease the number of Y1R-immunore-active (IR) cells and Y1R mRNA in the ARC [67]although, in the transgenic mice, we did not observe anyapparent change in the transcriptional activity of the Y1Rgene promoter in the ARC [430].

On the other hand, a change in the diet composition,with an increase in sugar intake, produces increased Y1R/LacZ transgene expression in both the ARC and thePVN of fed mice and in the ARC of 48 h-fasted mice

Table 1Changes in NPY mRNA, content and release and in Y1R gene expression induced by altered metabolic states

Region NPY mRNA NPY-IR or content NPY release Y1R mRNA Y1R/LacZ transgene

Hyperglycemia ARC �[63] — — �[67] �[430]PVN — — — — �[430]

Fasting ARC �[41,337] �[323] — �[67]�[422] () [430]PVN — �[56,323] �[87,172] — �[430]

Leptin treatment ARC �[336,366,379] () [388] �[366] — () [430]PVN — () [388] — — () [430]

Pregnancy ARC �[109] () [271] — �[271] �[271]PVN — �[271] — �[271] �[271]VMH — �[271] — �[271] �[271]

Diet-induced obesity ARC �[150] �[27,215] () [27,216] �[411] — () [149] () (Zammaretti et al,submitted for publication)

PVN — — — — () (Zammaretti et al,submitted for publication)

VMH �[124] — — — �(Zammaretti et al, submittedfor publication)

DMH �[124] — — — �(Zammaretti et al, submittedfor publication)

Leptin deficiency ARC �[329,366] �[35,162] — — () (Zammaretti et al,submitted for publication)

PVN — �[35] () [162] — — () (Zammaretti et al.,submitted for publication)

VMH — �[162] — — () (Zammaretti et al.,submitted for publication)

DMH — () [162] — — () (Zammaretti et al.,submitted for publication)

� = increase; � = decrease; () ¼ no change; — = not reported.

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[430].The majority of glucose sensitive neurons in the ARCwere shown to contain NPY [261]. Insulin-induced hypo-glycaemia was shown to increase NPY overflow in theDMH and in the PVN of control rats [120] and glucoseinjection decreases NPY protein expression in the ARC[63,350]. It can be hypothesize that the NPY signal in theARC/PVN axis might be dampened in response to hyper-glycaemic challenge, triggering the up-regulation of theY1R in ARC. Interestingly, the increase of Y1R/LacZ

transgene expression was observed in the rostral and midlevels of the ARC, a location where the percentage ofY1R-IR neurons that co express ACTH/POMC immuno-reactivity is highest [48]. This observation suggests that ina state of positive energetic balance, when the POMC prod-uct and other anorectic signals must be activated, the NPYinhibitory signal to POMC is also inhibited, triggering theup-regulation of Y1R in ARC.

Finally, the expression of Y1R/LacZ in the DMH andVMH was not altered to a significant extent by starvationor glucose administration, suggesting that other factorsmight be required to modulate the NPY-Y1R signallingwithin these nuclei (see below) [430].

A role for the Y1R in leptin deficiency has also been sug-gested. The increase in the hypothalamic NPY activityobserved in some rodent models of genetic obesity, whichcarry mutations at the leptin receptor level, is associatedwith a down-regulation of the Y1R [315]. For example,

obese Zucker rats show a diminished sensitivity to centralNPY administration [369] that is associated with thedecrease of NPY receptor density [236] as well as withthe decrease of Y1R mRNA expression in the hypothala-mus [22]. Moreover, the deletion of the Y1R reduces hyper-phagia and partially corrects the obese syndrome in ob/ob

mice [301]. We have recently observed that, although nosignificant changes in Y1R gene expression apparentlyoccur in the hypothalamus of ob/ob mice also expressingthe Y1R/LacZ transgene as compared to lean mice, leptinreplacement in these rodents increases Y1R/LacZ transgeneexpression both in the PVN and the DMH, suggesting anenhanced sensitivity of the NPY-Y1R pathway to theaction of leptin in ob/ob mice (Table 1) (F. Zammaretti,A. Luparia, P. Mele, G.C. Panzica, C. Eva, submitted forpublication).

On the other hand, some studies also showed thatrodent models of hypophagia and body weight loss canbe associated with both suppression of NPY signallingand a decrease of Y1R mRNA and immunoreactivity inthe hypothalamus. Treatments with cytokines, includinginterleukin2 and ciliary neurotrophic factor (CTNF), elicitanorexia by suppressing NPY-induced feeding and decreas-es NPY and Y1R mRNA expression in the medio-basalhypothalamus [421,422]. Mice with anorexia mutation(anx) are hypophagic, emaciated and exhibit NPY abnor-malities characterized by a dramatic decrease of NPY-IR

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terminal density in target areas, including the PVN, LH,DMH and ARC and in extra hypothalamic sites (forreview see [45]). These animals also display a significantreduction of Y1R mRNA in POMC positive neurons inthe ARC as well as in the PVN, suggesting that a decreaseof NPY/Y1R signal in the ARC/PVN axis might underliethe anx/anx phenotype [46].

Although the ARC-PVN axis is the hypothalamic path-way most involved in the NPY-Y1R mediated effects onfeeding behaviour, Y1R subtypes are also found in theDMH and the VMH [291]. Injections of NPY into therat VMH stimulates ingestive behaviour and this behaviouris enhanced in VMH lesioned rats and it is associated withthe up-regulation of Y1R mRNA [86,169,170]. On theother hand NPY in the DMH may be involved in feedingregulation mediated by reduced MC4R signalling, and anincrease in NPY mRNA in the DMH has been observedin some hyperphagic models in which hypothalamic mela-nocortin signalling is reduced, including lactation [219],diet induced obese mouse [124], MC4R null mice [66,187].

In diet-induced obesity models, when there is a positiveenergy balance, ARC NPY mRNA has been shown toincrease or decrease or remain unchanged and these com-pensatory changes apparently depend on the genetic back-ground of the different strains of rodents and on the type oftreatment [27,150,212,215,216,411]. Moreover, no signifi-cant changes of Y1R mRNA expression were found inthe ARC of obesity prone and obesity resistant mice fedwith a high fat diet, suggesting that the ARC NPY-Y1Rsystem does not play a prominent role in driving the hyper-phagia associated with palatable diet [149].

Conversely, a profound increase in the expression ofNPY has been found in the DMH and VMH of diet-in-duced obese mice, suggesting that the increased activityof NPY neurons in these brain regions might participatein the maintenance of energy homeostasis [124]. Indeed,increased neuronal activity in the DMH was found to con-tribute to the development of obesity caused by ingestionof high fat diet, whereas lesions of rat DMH attenuate highfat diet-induced weight gain [24]. Finally, mice susceptibleto diet-induced obesity have increased c-fos expression inthe DMH when fed with high fat diet [420].

In line with these observations, we have recently demon-strated that consumption of a high fat diet significantlydecreases Y1R gene expression in the DMH and VMH ofY1R/LacZ transgenic mice, and that leptin administrationfails to prevent this effect (Table 1) (F. Zammaretti, G.C.Panzica, C. Eva, submitted for publication). Conversely,Y1R gene expression in the ARC and PVN was not alteredin this obesity model. Y1R/LacZ transgenic mice were cre-ated and maintained on a FVB/n background, a mousestrain that develops diet-induced obesity and leptin resis-tance. Thus, the development of leptin resistance in highfat diet-fed Y1R/LacZ mice could compromise leptindependent regulation of the NPY-Y1R system in theserodents, making Y1R in the DMH and VMH partiallyresponsible for diet-induced weight gain. Previous studies

have shown a gender-related difference of the FVB mousestrain in leptin responsiveness [130]. Interestingly, weobserved that the modulation of DMH and VMH Y1Rgene expression in response to high fat diet is also sex relat-ed since the consumption of high fat diet failed to affectbody weight as well as Y1R gene expression in the VMHand DMH of Y1R/LacZ transgenic female mice (F. Zam-maretti, G.C. Panzica, C. Eva, submitted for publication).

Pregnancy is associated with a positive energy balanceprimarily due to an increase in food intake to preventdepletion of maternal energy stores. During pregnancy,both circulating leptin concentrations and food intake areelevated, suggesting that pregnancy may be a physiologicalstate of leptin resistance in the hypothalamus similar to theleptin resistance in obesity [10,233]. We demonstrated thaton day 18 of pregnancy there are no changes in NPY-IRneurons in the ARC, but a significant decrease in thePVN (Table 1). This last change is associated with theup-regulation of Y1R mRNA endogenous levels andY1R/LacZ transgene expression, suggesting that thesechanges may possibly occur through the activation of tran-scriptional mechanisms [271]. Conversely we observed aprofound induction of NPY immunoreactivity in theVMH of pregnant mice that in turn down-regulates Y1Rgene expression [271]. The increase of NPY-Y1R signallingin the VMH of pregnant mice can be interpreted as a com-pensatory response to the prolonged decrease of NPY inthe PVN, contributing to the hyperphagia observed duringpregnancy. The VMH is a key nucleus for regulating neu-roendocrine and behavioural functions during pregnancyand after parturition and contains estrogen receptors[384]. Thus, a direct effect of steroids on the Y1R geneexpression in the VMH cannot be ruled out. However thepossibility that the decrease of Y1R gene expression isdue to homologous down regulation appears more likely,given that it is associated with an increase in the densityof NPY-IR fibres.

NPY-Y1R signalling in the VMH may also play a role ininsulin secretion, and the central effect of glucocorticoids incontrolling insulin release appears to be mediated throughthe alteration of NPY-regulated pathways in this nucleus.Excessive corticosterone promotes body fat gain andhyperinsulinemia and also increases NPY synthesis andY1R mRNA expression in the ARC [2,208]. Converselyadrenalectomy, that reduces basal plasma insulin levels,abolishes the NPY stimulation of insulin release and induc-es the down regulation of Y1R selectively in the VMH[415].

5. Y1R and reproduction

A great amount of evidence supports the hypothesis thatNPY, acting at all levels of the hypothalamic-pituitary-go-nadal axis, is a crucial neuronal modulator of severalreproductive functions, including the generation of GnRHpulses and LH hormone surges, metabolic regulation ofreproduction, puberty, and sexual behaviour. These effects

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depend on the steroid hormone environment, levels ofNPY secretion, and the specific brain area.

NPY exerts a facilitatory role in the generation of pre-ovulatory GnRH and gonadotropin surges [77,78,171]. Inthe brain, NPY neurons, mainly located in the ARC andDMH hypothalamic nuclei and in the brainstem, senddirect projections to the preoptic area, ARC and medianeminence [331,385], where they may stimulate the preovu-latory surge of GnRH by either pre- or postsynaptic mech-anisms [30,78,386,419]. On the other hand, NPY can besecreted into the hypophysial-portal circulation[371,399,419] and it modulates the response of gonado-trope cells to the stimulatory action of GnRH by amplify-ing its capacity to trigger the LH preovulatory surge[18,77,371]. In addition, the ability of NPY to elicit GnRHrelease and LH surge is increased immediately before proe-strous [30]. This is supported by the demonstration thathypothalamic NPY gene expression [20,320], peptide levels[325] and release [399] increase concomitantly with sponta-neous or steroid-induced GnRH surges, and that systemicor central administration of anti-NPY antiserum[371,400] or NPY antisense oligodeoxynucleotides[168,423] result in the suppression of proestrous LH surgein steroid primed ovariectomized rats. Furthermore, theLH surge is significantly decreased in NPY knockout mice[424].

The effect of NPY on the GnRH secretion cannot occurwithout a proestrous hormonal environment. In numerousspecies, NPY has a negative effect on GnRH levels, pulseamplitude and frequency when levels of estrogen and pro-gesterone are low, as unprimed gonadectomized animals[171,188,239] and this effect is reverted by steroid replace-ment [171,188].

Growing evidence also suggests that NPY may at leastin part mediate communication among energy balance,GnRH secretion and sexual behaviour in a large numberof species, as diverse as rodents, fish and snails[4,258,265]. NPY consistently suppresses LH release whenadministered to ovariectomized animals or to intact ani-mals on a chronic basis, leading to the cessation of repro-duction [61,298]. NPY synthesis and release are greatlyincreased in response to metabolic challenge, such as star-vation and increased energy expenditure, that inhibit thepulsatile mode of LH secretion [172,218], and NPY levelsare reduced by treatments that ameliorate metabolic deficitand reinstate HPG function [174].

NPY treatment of male rats shifts attention toward eat-ing and away from sex [4,300], regardless of the presence ofa sexually receptive female, without affecting erectile orejaculatory functions [70,175]. NPY agonists increase foodintake and decrease lordosis duration in ovariectomizedSyrian hamsters brought into oestrous with ovarian steroidtreatment [74].

In addition, the reproductive effects induced by leptinsignal depletion may be partially caused by NPY hyperse-cretion [142,301,302]. For instance, NPY levels are elevatedin infertile ob/ob mice and Zucker fa/fa rats, and leptin

administration decreases ARC NPY expression and res-cues LH hormone levels and ovarian weight in ob/ob mice[65]. In addition, NPY hypersecretion inhibits sexualbehaviour in obese Zucker female rats, as demonstratedby the observation that NPY immunoneutralization atten-uates food intake and body weight gain, and reinstates sex-ual behaviour in these animals [229].

Recent evidence also suggests that NPY is a componentof the prepubertal brake on GnRH release and that areduction in inhibitory NPY signalling may initiate puber-ty. In support to this hypothesis, central NPY infusion inprepubertal rats induces a delay of puberty similar to thatinduced by food restriction [129,297], whereas the adminis-tration of a Y1R antagonist stimulates precocious GnRHsecretion [96]. Moreover, an inverse relationship betweenGnRH and NPY exists during the prepubertal–pubertaltransition, and ARC NPY mRNA expression is significant-ly higher in prepubertal juvenile male monkeys as com-pares to pubertal males [298]. However, sex differencesmay exist in the central mechanism controlling infantile-ju-venile transition. The inverse relationship between GnRHand NPY release was not found in female rhesus monkeys[119]. Likewise, infusion of NPY in the median eminencestimulates GnRH release in pubertal but not in prepubertalfemale monkeys, and NPY immunoneutralization fails tostimulate GnRH secretion in prepubertal female monkeys.

Pharmacological studies have shown that the stimulato-ry and inhibitory effects of NPY on GnRH secretion aremediated by different receptors. Y1R has been implicatedin the augmentation of LH release [161,173,214]. Numer-ous GnRH nerve terminals and fibers are co-localized withY1R positive staining both in the organum vasculosum andthe median eminence, and Y1R-positive fibers were foundin close apposition to GnRH cell bodies in the preopticarea, providing morphological bases for a physiologicalimportance of this receptor subtype [214,220,221]. Y1RmRNAs and NPY-IR cells are also expressed in the anteri-or pituitary tissue and in gonadotrope-enriched pituitarycell cultures [91,143]. Demonstration that NPY actsthought the Y1R subtype to stimulate LH preovulatorysurge first drawn from the observation that NPY and[Leu31Pro34]NPY similarly stimulate LH release and thata specific pharmacological blockade of the Y1R with selec-tive antagonists BIBP32226 and 1229U91 attenuates boththe LH surge in proestrous rats and surges induced byGnRH and NPY in pentobarbital-blocked proestrous rats[31,161,214,308]. NPY-induced augmentation of GnRHrelease during proestrous involves a dramatic increase oftissue responsiveness to NPY and this effect requires aproestrous hormonal environment [30]. Levine and co-au-thors [143,425] have shown that the influence of a steroidenvironment may affect Y1R-mediated signalling and thatestrogens up-regulate responsiveness to NPY through reg-ulation of Y1R gene expression in both the pituitary andthe hypothalamus. This was demonstrated by the observa-tion that Y1R mRNA expression is significantly increasedin the hypothalamus of proestrous or steroid-primed

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ovariectomized rats and that a similar up-regulation can beinduced by exogenous estrogen treatment. Furthermore,the increase of Y1R mRNA occurred only in conditionsin which GnRH responsiveness to NPY is enhanced, sinceit was not observed in unprimed ovariectomized rats. Thisregulatory mechanism appears to be operative also ingonadotropes since estrogen treatment of gonadotrope-en-riched pituitary cells increases their responsiveness to NPYand up-regulates Y1R mRNA expression. In addition it hasbeen suggested that the action of estrogens is mediated bythe induction and/or activation of progesterone receptorssince the antagonism of progesterone blocks the increaseof Y1R mRNA as well as the augmentation of tissueresponsiveness to NPY [19,425].

The mechanism whereby estrogens increase Y1R geneexpression may occur at the level of transcription. By tran-siently transfection assays we have demonstrated that 17b-estradiol increases Y1R gene promoter transcriptionalactivity and that this effect requires the presence of estrogenreceptor-a and it is inhibited by co expression of estrogenreceptor-b [263] (see above and Fig. 2A). A large numberof pituitary cells expresses estrogen receptors [253]. More-over, although a very low number of NPY-positive affer-ents to GnRH cell bodies co-express ERa [349], estrogensmight increase the expression of Y1R located on ERa-IRafferents to GnRH perikarya. Accordingly, the Y1R[209,220] and ER-a expression has been reported to co-lo-calize in the same regions within the ARC and adjacentregions of the mediobasal hypothalamus [280,347].

The inhibitory effects of NPY on LH secretion and sex-ual behaviour were first proposed to be mediated by Y5Rsince agonists that bind this receptor subtype inhibit LHrelease whereas Y5R antagonists prevent NPY inhibitionof LH surges [309].

However, increasing evidence suggests that the Y1R sub-type also mediates NPY-induced inhibition of the gonado-trope axis. The central administration of a Y1R antagonistto juvenile animals was shown to stimulate LH release andto elicit precocious GnRH release [96,302]. Moreover,Mills and co-authors [250] have suggested that the activa-tion of ARC Y1R inhibits sexual behaviour through theactivation of l-opioid receptor in the medial preoptic area.This effect is inhibited by the selective Y1R antagonist(Cys31, Nva34) NPY (27–36) and is under the stimulatoryor the inhibitory control of both estrogen and progester-one, respectively.

Furthermore, studies performed on Y1R knockout micesuggested a physiological role for the NPY-Y1R pathwayin the adaptation of gonadotrope axis activity to metabolicchanges. Adult Y1R deficient mice show an increased resis-tance to the fasting-induced inhibition of the gonadotropinaxis that is associated with increased circulating leptin lev-els [302]. In addition, the depletion of the Y1R restores nor-mal pituitary LH content and seminal vesicle weight in ob/ob mice, suggesting the existence of NPY effects indepen-dent on leptin action [302]. What is more, a recent studyhas shown that in juvenile mice lacking the Y1R, starvation

fails to induce the expected delay of puberty despite thedecrease of circulating leptin levels and the increase ofNPY mRNA expression in the hypothalamus [118].

6. Y1R in anxiety and stress

A large number of studies have demonstrated that a dis-tributed network of NPY containing elements, with theAmy and the hippocampus as core components, plays animportant role in the regulation of emotional behaviourand responsiveness to stressful stimuli (Fig. 3C). Intra-amygdalar or ICV administration of NPY produces apotent anxiolytic effect in several behavioural models ofanxiety [49,136,138,328] and NPY knockout mice exhibitan anxiogenic-like phenotype [12]. Moreover intra-amy-gdalar injection of a viral vector encoding NPY inducesan increase of NPY mRNA and peptide levels in theAmy that is associated with decreased anxiety relatedbehaviours in the elevated plus maze [304].

NPY inhibits several metabolic and behaviouralresponses to stress, including the adverse gastrointestinalconsequences, the anxious behaviour and the stress-in-duced decrease in pentobarbital-induced sleep time[12,49,136,138,180,328,381]. A functional antagonismbetween NPY and the corticotropin-releasing factor(CRF) has been also demonstrated in various nuclei alongthe stress/anxiety circuits such as the hippocampus [381],the hypothalamus [131], the locus coeruleus [64], the peri-aqueductal grey [64,184] and the septal complex [180].NPY inhibits the anxiogenic-like effect of CRF[42,64,135,180,182,381], suggesting that this neuropeptidemay act as an endogenous agent to buffer against the stress-or-induced release of CRF in the Amy, a structure longknown to be critical for the generation, expression andmaintenance of the stress/fear/anxiety in both experimentalanimals and humans [80,313,406]. In addition, NPY hasbeen shown to play a neuromodulatory effect on the hypo-thalamic-pituitary adrenal axis (HPA) [208,396,398].

Agonist, antagonist and antisense oligonucleotide stud-ies consistently indicate that the anxiolytic and the anti-stress actions of NPY are mediated through the activationof the Y1R. The involvement of the Y1R in the anxiolyticeffect of NPY was first suggested by the observation that[Leu31Pro34]NPY, administered ICV or directly into theCeA, elicits behavioural responses in several animal modelsof anxiety, including the elevated plus maze, the Vogel test,the conflict test and social interaction [43,49,133,181,328].Conversely, antisense oligonucleotides targeting the Y1R,which decrease the density of Y1R, induce behaviouraleffects on the elevated plus maze opposite to those seenafter NPY administration [133,394]. These studies werefurther confirmed by the observation that central adminis-tration of the Y1R antagonist BIBP3226 elicits behaviouralsigns of anxiety in several behavioural tests[181,182,184,304] that are reverted by the administrationof diazepam. Furthermore, the injection into the Amy ofthe more selective Y1R antagonist BIBO3304 blocks the

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anxiolytic-like effects of NPY in the social interaction test[328] and increases defecation during exploratory behav-iour in the open field [179]. More recent studies have shownthat the depletion of the Y1R in genetically mutated micecan either increase or decrease anxiety, depending on thediurnal rhythms [178]. Paradoxically, restraint stress causesa decrease in anxiety in Y1R knockout mice, suggestingthat compensatory changes in the Y2R [416] might be atleast in part responsible for the altered phenotype of theseanimals.

Studies performed in our laboratories and others pro-vided further evidence that endogenous NPY signalling(in particular that mediated by the Y1R) is implicated inneuronal stress response since significant changes in NPYneurochemistry are induced by a variety of physical andpsychological stress, including restraint. Several studieshave shown that the expression of NPY mRNA and con-tent is dynamic and sensitive to acute and repeatedrestraint stress in numerous regions of the forebrain, ponsand medulla [200,226,227,380,383]. However, it is not clearhow the exposure to restraint may alter brain NPY expres-sion, since repeated or acute restraint produce increases,decreases or no changes in NPY mRNA and NPY peptidecontent that apparently depended on strain, region orstressor paradigm. One of these studies has shown thatexposure to acute restraint reduces NPY mRNA and pro-tein concentrations in the Amy and cortex of rats, suggest-ing that downregulation of endogenous NPY induced byacute exposure to restraint might account in part for theanxiogenic-like effect of the stressor [383]. In contrast, theobservation that exposure to repeated restraint, that leadsbehavioural and endocrinologic adaptation, up-regulatesNPY gene expression in the Amy and in the ARC, suggeststhat increased synthesis and release of NPY may contributeto the development of habituation and that NPY may actto ‘‘buffer’’ the behavioural effects of stress-promoting sig-nals such as CRF [380]. Consistent with these observationswe showed that acute exposure to restraint increases Y1Rgene expression in the CeA, MeA and PVN of Y1R/LacZ

mice, suggesting that the up-regulation of the Y1R maybe a part of a compensatory mechanism triggered by thestress-induced reduction of functional NPY transmissionin the Amy (Fig. 4) [243]. Conversely repeated restraintfailed to affect Y1R/LacZ transgene expression in theAmy and PVN of mice, suggesting that a change in Y1Rgene promoter transcriptional activity is not required foradaptation to restraint stress [243]. Interestingly, the expo-sure to 5 min foot shock also failed to affect the Y1R intransgenic mice, suggesting that the modulation of theNPY-Y1R signalling requires longer-lasting stressful stimu-li (P. Mele, M. Serra, M. G. Pisu, G. Biggio, C. Eva,unpublished results) (Fig. 4).

7. Y1R and ethanol

NPY is also involved with neurobiological responses toethanol [284,375]. Voluntary alcohol consumption and

resistance to the intoxicating effects of ethanol are inverselyrelated to NPY levels in knockout and transgenic mice. Inparticular, NPY-null mutant mice consume significantlyhigher amounts of ethanol than do wild type strains,whereas transgenic mice over expressing NPY have a lowerpreference for ethanol than wild type mice [377].

Pharmacological studies suggest that central administra-tion of NPY decreases ethanol drinking in rodent linesselectively bred to have preference for ethanol over water.Central infusion of NPY decreases ethanol drinking andattenuates the severity of withdrawal symptoms in IndianaAlcohol Preferring rats (P) as compared to Indiana Alco-hol Non-preferring rats (NP) [8,115,116]. Similarly, ICVinfusion of NPY significantly reduces voluntary ethanolconsumption in High Alcohol Drinking (HAD) rats, show-ing high preference for ethanol, but does not alter ethanoldrinking in Low Alcohol Drinking (LAD) or outbred Wis-tar rats [7,116,185,186,352]. A recent study has shown thatNPY decreases ethanol intake in Wistar rats previouslyexposed to ethanol vapour for 8 weeks, a treatment thatincreases ethanol consummatory behaviour, further sug-gesting that the effects of NPY on ethanol intake are differ-ent in rodents when intake is moderate (i.e. outbred rats) orhigh (i.e. selectively bred alcohol preferring rats) [382].

The capability of NPY to decrease ethanol consumptionapparently is independent of its effect on feeding behav-iour. This was demonstrated by the observation that ICVadministration of NPY to HAD and LAD rats decreasesthe consumption of ethanol only in HAD rats, whereas itstimulates the intake of a 2.5% sucrose solution in bothHAD and LAD rats [7]. Moreover, NPY injected directlyin the PVN increases both ethanol consumption andsucrose intake in Long–Evans rats [186] and both ethanoland food intake in HAD and LAD rats [114], suggestingthat the effects of NPY in the PVN on alcohol intake arerelated to the caloric value of ethanol.

Rats that were selectively bred for alcohol preferencehave altered levels of NPY in several brain regions com-pared with alcohol-non-preferring rats, including theAmy, cingulate cortex, hypothalamus, hippocampus anddentate gyrus [55,92,132,157,289,372], with the most con-sistent finding that the decrease of NPY expression in theAmy can best explain their high alcohol drinking behav-iour. Moreover, Pandey and co-authors [289] have recentlyshown that NPY levels are decreased in the CeA, MeA of Prats when compared to NP rats, but not in the basolateralamygdala or in the core and the shell structures of nucleusaccumbens, suggesting that the neuronal substrate thatunderlays the NPY effect on alcohol abuse is probablythe extended Amy.

The results reviewed above suggest the possibility thatadministration of NPY reduces ethanol drinking only inanimal models with altered NPY signalling in the Amy.This hypothesis, however is not supported by the observa-tion that ICV infusion of NPY, at doses that potentiateethanol induced sedation, fails to affect ethanol intake ofCB57BL/6 mice [23,378], an inbred mouse strain that

Fig. 4. Upper panel: Effect of a single exposure to restraint stress or foot shock on Y1R gene expression in the medial amygdala (MeA), central amygdala(CeA) and paraventricular nucleus (PVN) of Y1R/LacZ transgenic mice. Adult male Y1R/LacZ mice from transgenic line 62 were subjected to 1 h ofrestraint stress or to 5 0 foot shock then killed 6 h after the end of the stress period. Shocks were given by a stimulator that delivered, for 0.5 s, every second,a shock of 0.1 mA. Modified from [243]. Lower panel: Effect of the treatment with finasteride, a selective inhibitor of 5-a-reductase, on the restraint stress-induced increase of b-galactosidase expression in the medial amygdala (A) or on the concentrations of 3a,5a tetrahydroprogesterone (3a,5a-TH PROG) inthe cerebral cortex (B) of Y1R/LacZ transgenic mice. Mice were treated for 2 days with vehicle (Veh, Restraint) or with 25 mg/kg of finasteride (Fin,Restraint + Fin) and killed 6 h (b-galactosidase) or immediately after the stressor (3a,5a-TH PROG). Coronal sections of the MeA, CeA, and PVN weresubjected to quantitative analysis of b-galactosidase histochemical staining and the density of transgene expression (dots/lm2) was determined aspreviously described [272]. Steroids were quantified by radioimmunoassay as previously described [342]. *p < 0.05 versus Veh (Newman–Keuls test).Modified from [243].

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shows high alcohol drinking behaviour [23,210,244,254,312] and low amygdalar NPY levels [132] comparedto the DBA/J2 mouse line.

Genetic evidence also suggests a possible role of NPY inethanol drinking behaviour. A quantitative trait loci anal-ysis conducted in F2 intercross progenies of P and NP ratsidentified a chromosomal region, that includes the NPYgene, that significantly correlates with differences in alcoholpreference between P and NP rats [33,58]; however, thiscorrelation was not confirmed in HAD and LAD rats[59,104,105].

The decrease of ethanol consumption elicited by NPYhas been suggested to be related to the pharmacologicactions of NPY. NPY produce electrophysiologicalresponses that are similar to those elicited by ethanol[93,94,353] and P and NP rats show opposite electrophysi-ological activity in the Amy following ICV infusion ofNPY [94].

It has been suggested that NPY may influence ethanolconsumption by modulating the sedative effect of this drug.NPY augments sedation induced by sodium pentobarbitaland ketamine [266,267,426], an effect that is mediated bythe activation of the Y1R, and ICV infusion of NPY poten-tiates the hypnotic-sedative effects of ethanol [376,378].Furthermore, low NPY signalling has been associated withincreased resistance to the sedative effect of ethanol, whileNPY over expression increases the sensitivity to ethanol-in-duced sedation [376,377].

Another possibility is that central infusion of NPYcould rescue ethanol drinking by reducing anxiety [7,367].Both human and animal studies have shown a strong asso-ciation between alcohol dependence and anxiety [245,284].P rats display higher baseline anxiety-like behaviours com-pared with NP rats, suggesting that they may drink exces-sive amounts of ethanol in order to reduce anxiety levels[367]. Phosphorylated CREB induces the expression of

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several downstream cAMP inducible genes, including NPY[62,237,287], and partial deletion of the CREB genedecreases NPY expression in the Amy [287]. Phosphory-lated CREB levels, similar to NPY expression levels, havebeen reported to be lower in the CeA and in MeA of P ratscompared to NP rats [157,252,286,288,427]. Additionally, asignificant reduction in CREB phosphorylation and NPYexpression were observed in rat amygdaloid and corticalstructures during ethanol withdrawal [285,286,316,431].Pandey and co-authors suggested that a decreased functionof CREB in the CeA and in MeA might regulate anxietyand alcohol intake via a decreased expression of NPY,and might provide a common link between anxiety andalcohol abuse [284,286,289]. Accordingly these authorshave recently showed that increasing the CREB phosphor-ylation–dependent NPY expression in the CeA and in MeAof P rats decreases anxiety levels and attenuates alcoholintake, whereas decreasing the CREB phosphorylation–de-pendent NPY expression in the CeA of NP rats increasesanxiety levels and alcohol preference [289]. Other dataare, however, inconsistent with this hypothesis. Forinstance, HAD rats have low amygdalar NPY levels andconsume large amount of alcohol but do not exhibit ananxiety like behaviour [7,157]. Similarly C57BL/6J mice,that consume higher amounts of ethanol and have lowNPY levels in the BLA and in the shell of the nucleusaccumbens, show lower basal level of anxiety when com-pared to DBA mice [122,205,251,279]. Moreover, transgen-ic mice over expressing NPY gene drink lower amounts ofethanol but do not have altered levels of anxiety as com-pared to wild type mice [377].

Several lines of evidence demonstrated that ethanoladministration and ethanol withdrawal influence NPYsignalling.

The effect of ethanol on NPY may vary with the dura-tion and type of ethanol treatment, rodent strain and brainregion. For instance, both investigator- and self-adminis-tered ethanol increases NPY levels in the hypothalamicstructures and in the ventrolateral medulla of Long–Evansrats [71]. Conversely no differences in NPY protein contentwere observed in several brain regions of Wistar rats fol-lowing a 7 week exposure to ethanol vapours [92], or inSprague Dawley rats fed for 15 days with an ethanol diet[316]. On the other hand, NPY immunoreactivity andNPY mRNA were shown to be decreased by single orrepeated administrations of intoxicating doses of ethanolin several brain regions, including the hypothalamus, hip-pocampus and cerebral cortex [36,192,316].

Conversely, consistent results have shown that NPY lev-els and mRNA decreased 24 h after withdrawal in somebrain regions that may be involved in seizure activity andanxious behaviour, including the cerebral cortex, hippo-campus and amygdaloid structures [36,316,431]. Thisdecrease is followed by a dramatic increase of NPY expres-sion in the hippocampus that occurs 72 h after withdrawal,that may represent a protective response against prolongedwithdrawal seizure activity [36,92].

The Y1R is located postsynaptically in several brainregions that are involved in the neurobiological responseto ethanol including the Amy and the hippocampus[269]. Pharmacological and genetic studies have implicat-ed the Y1R subtype in alcohol drinking behaviour,although conflicting results have been reported. Y1R-nullmutant mice maintained in C57BL/6 background con-sume higher amounts of solutions containing 3%, 6%,10% of ethanol, as compared to wild type mice, suggest-ing that the Y1R mediates the NPY-elicited decrease ofethanol consumption in these mice. Accordingly, micewith the deletion of the Y1R are also less sensitive tothe sedative effects of ethanol than wild type mice and thiseffect does not appear to be secondary to differences inacute clearance of ethanol [376]. In addition, Y1R knock-out mice show normal consumption of sucrose and qui-nine solutions, suggesting that the increased ethanoldrinking in these mice is not associated with altered tastepreference or energy homeostasis.

Conversely, pharmacological studies have demonstrat-ed that administration of Y1R antagonists decreases etha-nol consumption behaviour. Sparta et al. [361] haveshown that peripheral and central administration of theY1R antagonist [(-)-2-[1-(3-chloro-5-isopropyloxycarbon-ylaminophenyl)ethylamino]-6-[2-(5-ethyl-4-methyl-1,3-tia-zol-2-yl)ethyl]-4-morpholinopyridine] (compound A, alsonamed J-115814) [176] significantly reduces 8-h ethanolconsumption in C57BL/6J mice. These authors have sug-gested that this Y1R antagonist may reduce ethanoldrinking by acting on hypothalamic structures such asthe PVN, since the treatment with compound A signifi-cantly reduces short-term food intake by C57BL/6J mice.However, Schroeder and co-authors [332] have shownthat infusion of the selective Y1R antagonist BIBP3226into the Amy decreases self-administration by Long–Ev-ans rats, suggesting that other mechanisms may be oper-ative. Although the effect of BIBP3226 treatment ofLong–Evans rats on sedation has not been reported, thereduction of ethanol drinking caused by Compound Ain C57BL/6J mice does not seem to be related to the alter-ation of locomotor behaviour or the augmentation of eth-anol’s sedative effects, because intraperitoneal injection ofthis compound does not alter open-field locomotor activ-ity or ethanol-induced sedation [361]. On the other handthe possibility that the decrease of ethanol intake is relat-ed to the acute anxiogenic effects of the Y1R antagonistcannot be ruled out. Accordingly, it has been recentlyshown that ICV administration of both CRF and NPYinhibits ethanol intake in ethanol vapour-exposed Wistarrats, but when these peptides were given in combination,no differences from vehicle-treated rats were observed[382]. The effect of a long-term treatment with an Y1Rantagonist might help to explain the discrepanciesbetween pharmacological and genetic studies.

On the other hand, in spite of the key role that NPY sig-nalling may play in the modulation of voluntary alcoholconsumption and neurobiological responses to ethanol

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administration, only one study has investigated Y1R geneexpression in the brain of rodent lines bred for high orlow ethanol preference. Caberlotto et al. [55] have com-pared Y1R mRNA expression in several brain regions ofalcohol-preferring (Alko Alcohol rats, AA), alcohol-avoid-ing (Alko non-Alcohol rats, ANA) [255] and outbred Wis-tar rats. These studies showed that there were no significantchanges in Y1R mRNA expression in the cingulate cortex,hippocampus, CeA and MeA of ethanol naive rats fromAA, ANA or Wistar strains. However, in the same studythese authors have shown that these rat lines differ inregard to the alteration of NPY signalling from P andAP or HAD and LAD rats, since ICV infusion of NPYfailed to affect the alcohol-drinking behaviour of AA orANA rats and no significant differences in NPY levels werefound in the Amy of these rats.

8. Y1R and seizures activity

Several lines of evidence demonstrate that endogenousNPY is involved in modulating limbic seizure activity. Cen-tral administration of NPY decreases pharmacologicallyand electrically evoked seizures [354,392,418] and overexpression of NPY in transgenic mice reduces the severityof several types of seizures [390]. Seizures cause a signifi-cant up-regulation in NPY expression and release in hippo-campal neurons, and NPY-deficient mice exhibit increasedsensitivity to kainic acid-induced limbic seizures[13,311,390]. Pharmacological studies suggest that theY1R plays a permissive role in seizures, since the selectiveY1R antagonist BIBP3236 or Y1R antisense oligodeoxynu-cleotides significantly reduced seizure duration and numberand delays in kindling epileptogenesis, while the Y1R ago-nist [Leu32, Pro32]-PYY antagonizes this effect[26,110,391,392]. Moreover, intrahippocampal infusion ofthe Y1R antagonist BIBP3226 significantly reduces gener-alized tonic-clonic seizures induced by electroconvulsivestimulation [151]. The role of NPY-Y1R signalling in theregulation of limbic excitability is further supported by evi-dence of decreased Y1R expression in the epileptic tissue.Changes in Y1R expression have been reported in variousseizure models including electrically induced status epilep-ticus and kindling, [117,166,196]. In kainate-induced epi-lepsy a significant decrease of the Y1R binding andmRNA expression in the molecular layer of the dentategyrus and granule cells was observed [194]. In epilepticpatients with temporal lobe epilepsy, the Y1R was foundto have decreased in the dentate molecular layer of the hip-pocampus [107]. It was recently suggested that the Y1R islocalized on hilar NPY positive neurons and that thisreceptor subtype may act as an autoreceptor to inhibitNPY release during the high-frequency (epileptic)conditions under which it is released [290]. The significantdecrease of Y1R expression in epileptic tissues maytherefore represent a compensatory response to protectthe brain against recurrent seizures or neuronalhyperexcitability.

9. Y1R and depression

A large number of studies have indicated a role of NPYin depression. Centrally administered NPY elicits an anti-depressant-like effect in various animal models that havebeen shown to mimic, at least in part, a depression like phe-notype at the behavioural, biochemical and neurochemicallevel. In the forced swim test, an acute model widely usedas a screening test for potential antidepressant drugs,NPY was observed to decrease immobility time in adose-dependent manner, in both rats [310,368] and mice[310]. Furthermore, in olfactory bulbectomized rats, thatexhibit an acquired form of depressive-like behaviour, thesubchronic administration of NPY reduces hyperactivityin the open field, reverses the deficit in noradrenaline con-centrations in the Amy, reduces the concentrations of 5-HIAA in the hypothalamus, and improves lymphocyteproliferation [359].

On the other hand, NPY gene expression and peptidecontent were differentially altered in animal models ofdepression, depending on the region analyzed, althoughmost consistent changes were observed in the hippocam-pus. In particular, a significant decrease in NPY mRNAand content was found in the hippocampus of the Flinderssensitive line (FLS) rats, a genetic model of depression thatexhibits behavioural features characteristic of depression[281], when compared with the control Flinders resistantline (FLR) rats [53,54,155,156,163,165]. Interestingly,under basal conditions, NPY-like immunoreactivity waslower in female than in male FLS and FLR rats, which isin accordance with the higher prevalence of depression inwomen [165]. Likewise, in the Fawn-Hooded (FH) ratstrain, a genetic model of depression with a dysfunctionalserotoninergic system [282], hippocampal NPY mRNAand peptide content were decreased when compared toWistar or Sprague–Dawley rats [234].

In a maternal deprivation paradigm, that induces a phe-notype characterized by increased responsiveness of theHPA axis [299], NPY immunoreactivity was reduced inthe hippocampus and striatum while increased NPY levelswere observed in the hypothalamus [153,164]. Conversely,NPY mRNA and NPY immunoreactivity were increasedin the pyriform cortex and dentate gyrus of olfactory bul-bectomized rats [147,303]. More recently, in rats exposedto different behavioural tasks of depression, the stress-in-duced anhedonia model [341] and the learned helplessnessbehavioural test [159], NPY mRNA and NPY levels weresignificantly reduced in the hippocampal dentate gyrus.

Chronic treatment of experimental animals with clinical-ly effective antidepressants was shown to increase, decreaseor produce no changes on NPY mRNA and NPY proteincontent in various brain region including the hippocampus[53,54,154,155,408], the cerebral cortex [25,54,139,355,408]and the hypothalamus [25,53,54,139,155,227,408]. The lackof reproducibility of results might possibly depend on thedifferences in the experimental protocols rather than onthe mechanism of action of the drugs. Moreover, the

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pharmacokinetic of the different antidepressants as well asthe region-specificity of their effects make it difficult todraw any conclusion on their physiological significance(for review see [278]).

More consistent results were obtained followingrepeated, but not single, electroconvulsive shock (ECS)that is considered an animal model of electroconvulsivetherapy in humans. ECS increased NPY immunoreactivityand NPY mRNA in the hippocampus, cerebral cortex,hypothalamus [139,154,234,249,393,428]. Furthermore,ECS was found to increase extracellular levels of NPY infreely moving rats, suggesting that this treatmentstimulates both the synthesis and release of hippocampalNPY [152]. Similar results were obtained following treat-ment with lithium, a drug often employed for the treatmentand prevention of bipolar disorders, that increased NPYmRNA and NPY protein content in the hippocampus[153,154,235,402,429], nucleus accumbens [429], striatum[153,156,235,429], hypothalamus [153] and cerebralcortex [235].

Interestingly, the effect of antidepressants was signifi-cantly different in animal models of depression, whencompared with naive rats. In general, chronic treatmentwith antidepressant tends to normalize the decreasedNPY signalling in the hippocampus of animals with inher-ited depression behaviour. For instance, chronic treatmentwith fluoxetine, a selective inhibitor of serotonin reuptake,increases NPY mRNA and NPY immunoreactivity in thehippocampus of FLS rats, but it decreases NPY mRNAin FLR rats [53,54]. Likewise, ECS increases NPY mRNAin both FLS rats and FLR rats [163,165] and it induces alarger increase of NPY immunoreactivity in the hippo-campus of FH rats than in Wistar or Sprague–Dawleycontrol rats. In addition, subchronic treatment of learnedhelplessness rats with imipramine increased the number ofNPY-IR cells in the dentate gyrus but failed to produceany change in naive rats [159]. Finally, the treatment offluoxetine [53,54], lithium [153] and ECS [163,165]increased NPY mRNA and NPY immunoreactivity inthe hypothalamus of rats with inherited depression, sug-gesting that modulation of NPY hypothalamic signallingby antidepressant treatments might be involved in theirpharmacological efficacy on altered HPA homeostasis[278].

The involvement of the Y1R in the antidepressant actionof NPY was first demonstrated by pharmacological studiesshowing that ICV treatment with [Leu31;Pro34]PYYreduced the immobility time of rats in the forced swim test[368] and that the treatment with the selective Y1R antag-onists BIBP3226 and BIBO3304 blocked the anti-immobil-ity effect of NPY [310].

Few studies have investigated the effect of antidepres-sants on Y1R expression as compared to those establishedon its ligand. Caberlotto et al. [53,54] have shown thatchronic treatment with fluoxetine modulates, in a region-specific manner, Y1R expression in the brain of rats withinherited depression. In particular, fluoxetine increased

Y1R binding sites in the dentate gyrus, Amy and occipitalcortex of both FLS and FLR rats. Conversely, Y1RmRNA expression in the nucleus accumbens was reducedby fluoxetine in FLS rats but increased in FRL [53,54].In addition Masden et al. [224] have shown that the turn-over of the Y1R increases under electroconvulsive stimula-tions since repeated ECS stimuli increases Y1R mRNA anddecreases Y1R binding sites in the hippocampus of Wistarrats. These data suggest that the increase of hippocampalNPY signalling induced by antidepressant treatments inFLS (fluoxetine) or naive rats (ECS) might be reached byboth stimulating NPY synthesis (see above) and increasingY1R responsiveness.

10. Y1R and GABAergic transmission

Pharmacological and functional studies reviewedabove suggest that NPY have similar properties to thoseobserved with positive modulators of the GABAA recep-tor complex, like benzodiazepines and neuroactive ste-roids, on sleep [95,137,266,426], anxiety, stress[12,49,136,138,180,328,381], convulsions [14,97,249,418]as well as feeding behaviour [75,79,305,306,330].

GABA and NPY are co-expressed in the same neuronsin several brain regions, including the Amy [125,238,273],the hippocampus [167], the nucleus accumbens, the cere-bral cortex and the hypothalamus [148,306], and these neu-rotransmitters interact in the regulation of sedation,feeding, anxious behaviour and neuronal excitability[102,181,248,272,274,283,432]. For instance, intrahippo-campal administration of antisense oligonucleotidesagainst the c2 subunit of the GABAA receptor complex,that induces spontaneous electrographic hippocampal sei-zures and elevates threshold for seizures induced by electri-cal stimulation, increases NPY mRNA and NPY receptorbinding sites in several rat brain regions, particularly in thehippocampal region [248]. It has been suggested that theinduction of NPY is a part of a compensatory mechanismacting in response to a reduction in functional hippocam-pal postsynaptic GABAA receptors. Furthermore, acute,subchronic and chronic treatments with benzodiazepinesaffect the NPY immunoreactivity in the Amy, cerebral cor-tex and locus coeruleus and hypothalamus in naive rats[201] and in rats with conditioned fear produced in the pas-sive avoidance test [202], and these changes are dependenton the type of treatment and on the different brain nuclei.

A functional interaction between the GABAergic andNPY-Y1R mediated transmissions was first demonstratedby Kask and co-authors [181] that showed that the anxio-lytic benzodiazepine diazepam blocks the anxiogenic effectof Y1R antagonists. Subsequently, it was demonstratedthat NPY modulates sedation through the GABAergic sys-tem by interacting with the Y1R in the posterior hypothal-amus [267].

A recent study has shown that long-term treatment withflurazepam induces a decrease of NPY immunoreactivity inthe hippocampus of tolerant and dependent rats that is

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associated with the decrease of Y1R mRNA expression intolerant rats but not during withdrawal [432].

We extensively studied the interaction between GABAA

receptors and NPY-Y1R mediated transmissions in theAmy. By using Y1R/LacZ transgenic mice we showed thatGABA-IR neurons co-expressing NPY are scattered withinthe MeA, and in several cases they also bear the histochem-ical staining for Y1R/LacZ, suggesting that stimulation ofthe Y1R may regulate the activity of GABAergic neuronsin the MeA [273].

Furthermore, we observed that chronic modulation ofGABAA receptor function modulates Y1R gene expressionin the CeA and MeA of Y1R/LacZ transgenic mice. Thetreatment with positive (diazepam and abecarnil) or nega-tive modulators (FG7142) of GABAA receptor functioninduces, respectively, a significant increase, or decrease,of the Y1R gene expression in the MeA (Fig. 5) [272].Moreover, chronic treatment with neuroactive steroids,a-reduced derivatives of progesterone, such as 3a-hy-droxy-5a-pregnane-20-one (3a-5a-TH PROG) and 3a,5a-tetrahydrodeoxycorticosterone (3a,5a-TH DOC) thatact as positive modulators of GABAA receptor function[204,225] and elicit in vivo anxiolytic, anticonvulsant andhypnotic/anesthetic effects [37,195,397], induces an increaseof Y1R gene expression in the MeA of Y1R/LacZ transgen-ic mice similar to that induced by diazepam and abecarnil[102] (Fig. 5).

In rats, fluctuations in endogenous brain concentrationsof neurosteroids (during pregnancy, after delivery or dur-ing steroid treatment and withdrawal) can lead to behav-ioural changes and parallel alterations of GABAA

receptor subunit isoform gene expression [52,73,103,357].

Fig. 5. Effect of repeated treatment with positive and negative modulatorsof GABAA receptor on Y1R gene expression in the medial amygdala ofY1R/LacZ transgenic mice. Y1R/LacZ transgenic mice were treated for 15days with vehicle, i.p. (VEH), diazepam, 20 mg/kg i.p. (D20), abecarnil,6 mg/kg s.c. (ABE6) and FG 7142, 20 mg/kg i.p. (FG20) or for 10 dayswith 3a,5a-TH PROG 5 mg/kg, i.p. (allopregnanolone, ALLO5), thenkilled 90 min after the last injection. Data were analyzed as described inFig. 4 and are expressed as percentage of vehicle treated mice. *p < 0.05versus VEH (Newman–Keuls test). Modified from [102,272].

We demonstrated that the increase of progesterone plas-ma concentrations during pregnancy (Fig. 6C), or follow-ing long-term progesterone treatment (Fig. 6D), increasesY1R/LacZ transgene expression (Fig. 6A and B) as wellas Y1R mRNA in the MeA [274]. These effects are inhibit-ed by the treatment with finasteride, a selective blocker of5a-reductase that inhibits the synthesis of neuroactive ste-roids [6], suggesting that fluctuations in endogenous brainconcentrations of neuroactive steroids may affect boththe GABAA receptor and the Y1R function (Fig. 6A–D).

Increases in plasma and brain concentrations of neuros-teroids have been shown to be elicited by the exposure tovarious acute stress paradigms such as swim stress [307],foot shock stress [15,16] or restraint stress [243].

We therefore investigated whether the increase in Y1Rgene expression induced by acute restraint stress could bemediated by the short-term increase in the brain levels ofthe neuroactive steroids 3a,5a-TH PROG and 3a,5a-THDOC and the consequent actions of these steroids at theGABAA receptor. Our observations demonstrate that fin-asteride fails to prevent the increase of Y1R/LacZ trans-gene in the Amy and in the PVN induced by acuterestraint (Fig. 4A and B), suggesting that changes in thebrain concentrations of neuroactive steroids and in Y1Rgene expression are achieved independently of each other[243]. Additionally, the observation that repeated exposureto restraint, that induces repetitive and transient increasesin the concentrations of neuroactive steroids in the cerebralcortex, also fails to affect Y1R gene expression suggests thata sustained increase in the brain concentrations of thesesteroids may be required for the modulation of Y1R geneexpression. This conclusion is sustained by the evidencethat exposure for 5 min to foot shock stress also failed toaffect the Y1R in transgenic mice (Fig. 4).

The results reviewed above suggest that physiological orpathological conditions that induce a chronic modulationof GABAA receptor function may also affect Y1R function,possibly by triggering compensatory changes on NPY-con-taining neurons that, in turn, might be responsible for theup-regulation of the Y1R gene. The functional interactionbetween the GABAA receptor and NPY-Y1R mediatedpathways in the Amy might represent an important regula-tory mechanism for the modulation of emotionalbehaviour.

The interaction between GABAA, Y1R and neuroactivesteroids might also be operative in the regulation of neuro-endocrine stress response during late pregnancy.

In addition to its roles in parturition and lactation, oxy-tocin is a stress hormone in rats [206] and its secretion isenhanced in response to both emotional and physicalstressors [319]. NPY exerts its effects on the HPA axis byacting centrally at the level of the CRF neurons to evokeCRF release [126]. Similarly, it has been shown that NPYacts in the supraoptic nucleus (SON) to trigger oxytocinrelease into the general circulation [292]. The presence ofY1R-immunoreactivity on magnocellular neurons of PVNand SON and on parvocellular elements of PVN, that

Fig. 6. (A and C) Changes of b-galactosidase expression in the medial amygdala (A) and of 3a,5a tetrahydroprogesterone (3a,5a TH-PROG)concentrations in the cerebral cortex (C) of estrous and pregnant Y1R/LacZ mice, treated with vehicle or finasteride, and mice immediately after delivery.Vehicle or finasteride (25 mg/kg, s.c.) were injected daily from day 10 to day 17 of pregnancy, and mice were killed on day 18 (P18 and P18 + FIN,respectively). Estrous mice were similarly treated with vehicle (E) or with finasteride (FIN) for 7 days. Values are also shown for vehicle-treated mice 2days after delivery (PD2). Quantitative analysis of b-galactosidase and 3a,5a TH-PROG measurement were performed as in Fig. 4. *p < 0.05 versus E,FIN, P18 + FIN and PD2 (Newman–Keuls test). (B and D) Effect of long-term treatment with progesterone on b-galactosidase expression in the medialamygdala (B) and on 3a,5a-TH PROG concentrations in the cerebral cortex (D) of Y1R/LacZ mice. Progesterone treatment was performed according tothe 3-week, single-injection, multiple-withdrawal paradigm previously described [256]. Estrous mice were treated with vehicle (E), with progesterone (5 mg/kg, i.p) (PRO, PRO WD), or with progesterone and finasteride (PRO + FIN) and were killed 90 min (E, PRO, PRO + F) or 24 h (PRO WD) after the lastinjection (WD indicates withdrawal). Quantitative analysis of b-galactosidase and 3a,5a TH-PROG measurement were performed as in Fig. 4. *p < 0.05versus E, PRO WD, and PRO + FIN groups (Newman–Keuls test). Modified from [102,274].

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coexpress CRF and arginine vasopressin (AVP) understressful conditions, suggests that the activation of thisreceptor subtype may influence the synthesis and/or releaseof oxytocin, AVP and CRF [417].

Brunton and co-authors [50] have recently shown thatthe responsiveness of CRF/AVP and oxytocin neurons tocentral administration of NPY is attenuated in late preg-nant rats.

These authors suggested that suppression of NPY-stim-ulated oxytocin secretion and HPA axis activation in latepregnancy could be a result of an increased effectiveness

of GABAergic inhibitory input [50]. In particular, it hasbeen shown that the increase in the brain concentrationsof the neuroactive steroid 3a,5a-TH PROG during preg-nancy enhances the GABAergic inhibition of magnocellu-lar oxytocin neurons, possibly through its actions onGABAA receptors [51,319].

An interesting hypothesis is that the prolonged stimula-tion of GABAA receptors by 3a,5a-TH PROG duringpregnancy might alter Y1R expression in the SON orPVN, attenuating in this way the responses of the CRF/AVP and oxytocin neurons to NPY.

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11. Functional interaction between neuropeptide Y receptors

The results summarized in this review clearly indicatethat the Y1R plays an important role in a variety ofNPY-induced pathways. However, in the brain, the effectsof NPY can also be mediated by the activation of the Y2Rand Y5R subtypes, which are present in many regions.

The Y2R is abundantly expressed in the hippocampalformation and brainstem nuclei [89]. In the hippocampus,the Y2R is located on glutamatergic terminals where it sup-presses glutamatergic transmission through presynapticmechanisms [121,348]. Moreover, this receptor is also con-sidered to function as a presynaptic autoreceptor on NPY-ergic terminals where its activation inhibits the release ofendogenous NPY [190,191]. The involvement of the Y2Rin the regulation of emotionally related behaviour, withan anxiogenic effect upon activation, has been demonstrat-ed in several behavioural paradigms (for review see [60]).Furthermore, a pharmacological blockade as well as genet-ic inactivation of the Y2R mimics anxiolytic behaviouraleffects induced by Y1R-stimulation. The Y2R is also impli-cated in alcohol consumption [60]. The central administra-tion of selective Y2R antagonists decreases voluntaryethanol consumption and the phenotype of Y2R knockoutmice is analogous to the behaviour observed after injectionof Y2R antagonists in normal mice.

The exact role of the Y2R in feeding and energy regulationremains unknown. Central administration of Y2R agonistsreduces food intake in rodents [213]. However, hypothala-mus-specific Y2-deleted mice show a transient increase infood intake but a significant decrease in body weight, andgerm-line Y2R knockout mice are reported to have decreasedbody weight and food intake [327]. It has been recentlysuggested that the endogenous Y2R agonist PYY3-36 actspredominantly on the Y2R in the ARC of the hypothalamusto reduce food intake, but this observation has beenquestioned in several studies, as reviewed recently [38].

In situ hybridization signals of the Y5R mRNA are seenin a number of hypothalamic nuclei, the dentate gyrus andCA3 area of the hippocampus, cingulate cortex and in theAmy [89,112].

The Y5R has similar actions to the Y1R in that it stim-ulates food intake [231], elicits anxiolytic-like effects in sev-eral models of anxiety [60], causes sedation [360] andregulates GABA release [305].

Pharmacological and genetic evidence suggest that Y5R isalso involved in the anticonvulsant effect of NPY and that itplays a prominent role in inhibiting excitatory neurotrans-mission in mice, at least in the CA3 region. Moreover, theactivation of the Y5R contributes to the inhibitory actionsof NPY on reproductive hormone secretion [309].

Recent evidence suggested that different NPY receptorscan form homodimers and, possibly, heterodimers thatcould affect the expected physiology and pharmacologyof each receptor individually [28,84]. Silva and co-authors[348] have shown the existence of a physiological interac-tion between the Y1R and the Y2R and between the Y2R

and the Y5R in modulating stimulated-glutamate releasein the rat hippocampus and that, in conditions of co-activa-tion, the Y2R plays a predominant role. These authors pro-posed the elegant hypothesis of the formation of oligomersof the Y1R and the Y2R and of the Y2R and the Y5R andthat the activation of these complexes would predominant-ly activate the Y2R over the Y1R and the Y5R. Althoughfurther studies are required to determine the prevalenceand relevance of NPY receptor oligomers in native tissues,dimerization could have relevant implications for the phar-macology and physiology of these receptors.

12. Conclusions

The scarce availability of pharmacological tools as wellthe conflicting results provided by germ-line knockoutmouse models have made it difficult to demonstrate therole of NPY-Y1R signalling in physiology and disease.Nonetheless, numerous advances in our understanding ofY1R function have been made by studying tissue-specificchanges in its expression. Compensatory changes in Y1Rgene expression occur almost any time that NPY synthesisand release are increased or reduced by neuronal activity orperipheral hormones, suggesting that NPY-Y1R signallingis tightly regulated. Profound plastic changes of Y1R geneexpression are induced by positive or negative states ofenergy balance, reproductive activity and different kindsof brain injures, including prolonged stressful stimuli andepileptic seizures. These observations indicate that Y1R-mediated transmission is regulated by conditions that arecritical for survival, suggesting that NPY-Y1R signallingmay act to buffer against treating stimuli. The finding thatNPY and GABA exert similar effects on many behaviouraland physiological functions and that changes in GABAA

receptor-mediated signal also affect Y1R gene expressionsuggests that a functional interaction between GABAA

receptors and Y1R may play a pivotal role in maintainingenvironmental homeostasis.

Acknowledgments

This work was supported by grants 9905045782,2001055774 and 2003052254 to G. B and C.E. from theMinistero dell’Universita e della Ricerca Scientifica e Tec-nologica (Projects of National Relevance, Article 65DPR382/80), the grant, Progetti di ricerca sanitaria finalizzata2003-2005 from the Health Ministry of Regione Piemonte,the grant, Progetti di Ricerca scientifica applicata 2003-2005 from the Health Ministry of Regione Piemonte andby Telethon, project n. D.82 to C.E. F.Z. was supportedby a Telethon fellowship (project n.D.82 to C.E.).

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