The mPVN mediates blockade of NPY-induced feeding by a Y5 receptor antagonist: a c-FOS analysis

To identify the site(s) of NPY Y5 receptor (Y5R) mediation of NPY-induced feeding, we employed c-Fos immunostaining and a selective Y5R antagonist (Y5R-A), CGP71683A, in adult male rats. Intracerebroventricular (icv) administration of NPY stimulated feeding and c-Fos-like immunoreactivity (FLI) in the dorsomedial hypothalamus, supraoptic nucleus and the two subdivision of the hypothalamic paraventricular nucleus (pPVN), the parvocellular (pPVN), and magnocellular (mPVN). Y5R-A on its own, injected either intraperitoneally or icv, neither affected feeding nor FLI in hypothalamic sites. However, Y5R-A pretreatment suppressed NPY-induced food intake and FLI selectively in the mPVN. Taken together with our previous similar finding of Y1R involvement, these results suggest that NPY receptor sites concerned with feeding behavior reside selectively in the mPVN and Y1 and Y5 receptors are either coexpressed or expressed separately in those target neurons that promote appetitive drive.     

Nesfatin‐1, corticotropin‐releasing hormone,thyrotropin‐releasing hormone,and neuronal histamine interact in the hypothalamus to regulate feeding behavior

Nesfatin‐1, corticotropin‐releasing hormone (CRH), thyrotropin‐releasing hormone (TRH), and hypothalamic neuronal histamine act as anorexigenics in the hypothalamus. We examined interactions among nesfatin‐1, CRH, TRH, and histamine in the regulation of feeding behavior in rodents. We investigated whether the anorectic effect of nesfatin‐1, α‐fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), a CRH antagonist, or anti‐TRH antibody affects the anorectic effect of nesfatin‐1, whether nesfatin‐1 increases CRH and TRH contents and histamine turnover in the hypothalamus, and whether histamine increases nesfatin‐1 content in the hypothalamus. We also investigated whether nesfatin‐1 decreases food intake in mice with targeted disruption of the histamine H1 receptor (H1KO mice) and if the H1 receptor (H1‐R) co‐localizes in nesfatin‐1 neurons. Nesfatin‐1‐suppressed feeding was partially attenuated in rats administered with FMH, a CRH antagonist, or anti‐TRH antibody, and in H1KO mice. Nesfatin‐1 increased CRH and TRH levels and histamine turnover, whereas histamine increased nesfatin‐1 in the hypothalamus. Immunohistochemical analysis revealed H1‐R expression on nesfatin‐1 neurons in the paraventricular nucleus of the hypothalamus. These results indicate that CRH, TRH, and hypothalamic neuronal histamine mediate the suppressive effects of nesfatin‐1 on feeding behavior.     

Endogenous GLP-1 acts on paraventricular nucleus to suppress feeding: Projection from nucleus tractus solitarius and activation of corticotropin-releasing hormone,nesfatin-1 and oxytocin neurons

Glucagon-like peptide-1 (GLP-1) receptor agonists have been used to treat type 2 diabetic patients and shown to reduce food intake and body weight. The anorexigenic effects of GLP-1 and GLP-1 receptor agonists are thought to be mediated primarily via the hypothalamic paraventricular nucleus (PVN). GLP-1, an intestinal hormone, is also localized in the nucleus tractus solitarius (NTS) of the brain stem. However, the role of endogenous GLP-1, particularly that in the NTS neurons, in feeding regulation remains to be established. The present study examined whether the NTS GLP-1 neurons project to PVN and whether the endogenous GLP-1 acts on PVN to restrict feeding. Intra-PVN injection of GLP-1 receptor antagonist exendin (9–39) increased food intake. Injection of retrograde tracer into PVN combined with immunohistochemistry for GLP-1 in NTS revealed direct projection of NTS GLP-1 neurons to PVN. Moreover, GLP-1 evoked Ca²⁺ signaling in single neurons isolated from PVN. The majority of GLP-1-responsive neurons were immunoreactive predominantly to corticotropin-releasing hormone (CRH) and nesfatin-1, and less frequently to oxytocin. These results indicate that endogenous GLP-1 targets PVN to restrict feeding behavior, in which the projection from NTS GLP-1 neurons and activation of CRH and nesfatin-1 neurons might be implicated. This study reveals a neuronal basis for the anorexigenic effect of endogenous GLP-1 in the brain.     

Brain‐derived neurotrophic factor,corticotropin‐releasing factor,and hypothalamic neuronal histamine interact to regulate feeding behavior

Brain‐derived neurotrophic factor (BDNF), corticotropin‐releasing factor (CRF), and hypothalamic neuronal histamine are anorexigenic substances within the hypothalamus. This study examined the interactions among BDNF, CRF, and histamine during the regulation of feeding behavior in rodents. Food intake was measured after treatment with BDNF, α‐fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), or CRF antagonist. We measured food intake in wild‐type mice and mice with targeted disruption of the histamine H1 receptor (H1KO mice) after central BDNF infusion. Furthermore, we investigated CRF content and histamine turnover in the hypothalamus after BDNF treatment, and conversely, BDNF content in the hypothalamus after histamine treatment. We used immunohistochemical staining for histamine H1 receptors (H1‐R) in BDNF neurons. BDNF‐induced feeding suppression was partially attenuated in rats pre‐treated with FMH or a CRF antagonist, and in H1KO mice. BDNF treatment increased CRF content and histamine turnover in the hypothalamus. Histamine increased BDNF content in the hypothalamus. Immunohistochemical analysis revealed that H1‐Rs were expressed on BDNF neurons in the ventromedial nucleus of the hypothalamus. These results indicate that CRF and hypothalamic neuronal histamine mediate the suppressive effects of BDNF on feeding behavior and body weight.     

兔脑内Orexin B免疫阳性神经元的分布定位

采用免疫组织化学方法研究了10只青紫蓝兔脑内Orexin B免疫阳性神经元的分布定位。结果显示,Orexin B免疫阳性神经元分布于下丘脑的室旁核、背内侧核、穹隆周核、外侧区和后区以及底丘脑的未定带。以下丘脑背内侧核、穹隆周核和外侧区的阳性神经元数量较多,下丘脑室旁核、后区和未定带较少。表明了兔脑内Orexin B免疫阳性神经元的分布与Orexin A免疫阳性神经元的分布存在一些差异,提示两种Orexin的产生部位和生理功能可能也存在差异。     

Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens

Ghrelin, a powerful orexigenic peptide released from the gut, stimulates feeding when injected centrally and has thus far been implicated in regulation of metabolic, rather than hedonic, feeding behavior. Although ghrelin's effects are partially mediated at the hypothalamic arcuate nucleus, via activation of neurons that co-express neuropeptide Y and agouti-related protein (NPY/Agrp neurons), the ghrelin receptor is expressed also in other brain sites. One of these is the ventral tegmental area (VTA), a primary node of the mesolimbic reward pathway, which sends dopaminergic projections to the nucleus accumbens (Acb), among other sites. We injected saline or three doses of ghrelin (0, 0.003, 0.03, or 0.3 nmol) into the VTA or Acb of rats. We found a robust feeding response with VTA injection of ghrelin, and a more moderate response with Acb injection. Because opioids modulate feeding in the VTA and Acb, we hypothesized that ghrelin's effects in one site were dependent on opioid signaling in the opposite site. The general opioid antagonist, naltrexone (NTX), injected into the Acb did not affect feeding elicited by ghrelin injection into the VTA, and NTX in the VTA did not affect feeding elicited by ghrelin injected into the Acb. These results suggest interaction of a metabolic factor with the reward system in feeding behavior, indicating that hedonic responses can be modulated by homeostatic factors.     

Neuropeptide FF2 receptor distribution in the human brain. An immunohistochemical study

Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Hypothalamic galanin is up-regulated during hyperphagia and increased body weight gain induced by disruption of signaling in the ventromedial nucleus

Disruption of signaling in the ventromedial nucleus (VMN) by colchicine (COL) produces transient (4 days) hyperphagia and weight gain. Microinjection of galanin into various hypothalamic sites stimulates feeding, so we tested the hypothesis that galanin is up-regulated in COL-treated rats by analyzing galanin concentrations in micropunched hypothalamic sites. Galanin was increased in the paraventricular nucleus on Days 1 through 4 after COL-injection. Galanin was also elevated in three other hypothalamic sites, the dorsomedial nucleus, lateral hypothalamic area, and perifornical hypothalamus, on Days 2-4 and in the lateral preoptic area, on Day 1 only. In the median eminence-arcuate nucleus and amygdala an initial decrease on Day 1 was followed by a then progressive increase through Day 4. These increases occurred despite marked elevations in blood insulin and leptin, hormones known to suppress hypothalamic galanin. When COL- or saline-treated rats were injected intracerebroventricularly with galanin, it stimulated feeding further in the hyperphagic COL-treated rats, but the relative response over basal consumption was similar in both COL-treated and control rats. These results in VMN disrupted rats suggest that neurochemical rearrangements, including increased availability of galanin, may contribute to the hyperphagia and increased weight gain; additionally, it seems that neurons in the VMN normally exert a restraint on galanin signaling.     

Distribution of neurons containing leptin receptors in the hypothalamus of the pig

Leptin, secreted by white adipocytes, has profound feeding, metabolic, and neuroendocrine effects. Leptin acts on the brain, but specific anatomical sites and pathways responsible for mediating these effects are still unclear. We have systematically examined the distribution of leptin receptor containing neurons in the porcine hypothalamus by means of immunohistochemical staining methods. Leptin receptor immunoreactivity (OBR-IR) was observed in both the preoptic area and anterior hypothalamic area. No immunoreactive structures were found in the median eminence. Only single, small neurons were observed in the arcuate nucleus. The most abundant OBR-IR cell bodies were located in the supraoptic nucleus. In the paraventricular nucleus, OBR-IR neurons were moderate in number. Single, dispersed neurons were found in the ventromedial nucleus. These findings indicate that there are distinct OBR-IR neuronal populations in the porcine hypothalamus and leptin not only plays an integrative role in feeding behavior, but also in neuroendocrine activity.     

Peripheral ghrelin participates in the glucostatic signaling mediated by the ventromedial and lateral hypothalamus neurons

Employing immunohistochemistry techniques, we examined the c-fos expression in different hypothalamic areas, when plasma glucose levels were modified by the administration of insulin and 2-deoxyglucose (2-DG) respectively. Subsequently, the hypoglycemia produced by an injection of insulin significantly increased feeding concomitant to higher c-fos expression in the arcuate nucleus (ARC), paraventricular nucleus (PVN), dorsomedial hypothalamus (DMH) and lateral hypothalamus (LH), while no statistical changes in the ventromedial hypothalamus (VMH) were found. Also, the glucopenia induced by 2-DG administration produced similar stimulatory effects on appetite and the neuronal activity affecting all the hypothalamic areas studied, including the VMH. The peripheral blockade of the orexigenic hormone ghrelin with a specific antibody (AGA) significantly decreased food intake as induced from acute hypoglycemia and glucopenia. Curiously, the conjoint AGA and insulin or 2-DG administration produced a differential effect on the hypothalamic neurons analyzed, by increasing the number of c-fos positive neurons in the ARC, PVN and DMH, but not in the VMH and LH. This outcome suggests an interactive effect of the glucostatic pathways involving these two areas with the ghrelin signaling.     

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.     

Angiotensin II in dorsomedial hypothalamus modulates cardiovascular arousal caused by stress but not feeding in rabbits

The dorsomedial hypothalamus (DMH) is critically implicated in the cardiovascular response to emotional stress. This study aimed to determine whether the DMH is also important in cardiovascular arousal associated with appetitive feeding behavior and, if so, whether locally released angiotensin II and glutamate are important in this arousal. Emotional (air-jet) stress and feeding elicited similar tachycardic (+51 and +45 beats/min, respectively) and pressor (+16 and +9 mmHg, respectively) responses in conscious rabbits. Bilateral microinjection of GABA(A) agonist muscimol (500 pmol) into the DMH, but not nearby hypothalamic regions, attenuated pressor and tachycardic responses to air-jet by 56-63% and evoked anorexia. Conversely, stimulation of the DMH with the glutamate analog kainic acid (250 pmol) elicited hypertension (+25 mmHg) and tachycardia (+114 beats/min) and activated feeding behavior. Local microinjection of a glutamate receptor antagonist, kynurenic acid (10 nmol), decreased pressor responses to stress and eating by 46 and 72%, respectively, without affecting feeding behavior. Bilateral microinjection of a selective AT(1)-receptor antagonist, candesartan (500 pmol), into the DMH, but not nearby sites, attenuated pressor and tachycardic stress responses by 31 and 33%, respectively. Candesartan did not alter feeding behavior or circulatory response to feeding. These results indicate that, in addition to its role in mediating stress responses, the DMH may be important in regulating cardiovascular arousal associated with feeding. Local glutamatergic inputs appear to regulate cardiovascular response to both stress and feeding. Conversely, angiotensin II, acting via AT1 receptors, may selectively modulate, in the DMH, cardiovascular response to stress, but not feeding.     

Colocalization of Glucagon-Like Peptide-1 (GLP-1) Receptors, Glucose Transporter GLUT-2, and Glucokinase mRNAs in Rat Hypothalamic Cells: Evidence for a Role of GLP-1 Receptor Agonists as an Inhibitory Signal for Food and Water Intake

Abstract: This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic β cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7–36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7–36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1–37), a peptide with lower biological activity than GLP-1 (7–36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7–36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9–39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7–36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7–36) amide may modulate both food and drink intake in the rat through a central mechanism.     

Diurnal rhythm of alpha 2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior

The paraventricular nucleus alpha 2-noradrenergic system and the glucocorticoid hormone, corticosterone, are known to modulate feeding behavior and exhibit a circadian pattern which may be related to the natural periodicity of feeding in the rat. The results of the present study indicate that the binding of [3H]p-aminoclonidine to alpha 2-noradrenergic receptors specifically in the paraventricular nucleus varies concomitantly with plasma corticosterone levels, as well as spontaneous feeding. A monophasic peak of paraventricular noradrenergic receptor binding is detected at the onset of the dark period, when corticosterone levels are highest and feeding is initiated. On the other hand, the supraoptic nucleus exhibits the reverse diurnal pattern, i.e., a significant decline of [3H]p-aminoclonidine binding at the onset of the dark period. Other hypothalamic and extra-hypothalamic areas fail to show significant changes in alpha 2-noradrenergic receptors as a function of the diurnal cycle. This study supports other evidence indicating a close interaction between circulating corticosterone and alpha 2-noradrenergic receptors in specific hypothalamic areas. It also reveals a potential importance for this interaction in control of the natural feeding rhythm.     

Ghrelin对大鼠摄食的影响及orexins信号通路的调控

目的:探究Ghrelin对大鼠摄食的影响及orexins信号通路的调控作用。方法:采用免疫组织化学染色的方法观察Ghrelin免疫阳性神经元轴突末梢与orexin神经元的突触联系以及下丘脑外侧区(LHA)内c-fos的表达。侧脑室注射抗-orexin-A IgG和抗-orexin-B IgG混合液、抗-黑色素浓集激素(MCH)IgG、NPY-1受体拮抗剂后测量大鼠摄食量,观察其对ghrelin诱导摄食的影响。结果:Ghrelin免疫阳性神经元轴突末梢与orexin神经元的突触相接触。侧脑室注射ghrelin可诱导orexin神经元内c-fos表达,但是没有引起MCH神经元内c-fos的表达。预先注射抗-NPY IgG抗体,ghrelin仍然可诱导orexin神经元内c-fos表达。侧脑室预先注射抗-orexin-A IgG和抗-orexin-B IgG抗体可减弱ghrelin促摄食作用,但是预先注射抗-MCH IgG抗体对ghrelin诱导的摄食作用没有明显影响。注射NPY受体拮抗剂可进一步加强抗-orexin-A IgG抗体和抗-orexin-B IgG抗体对ghrelin诱导摄食的抑制效应。结论:ghrelin可能与orexin系统相互作用共同参与摄食和能量平衡的调控。     

An eGFP-expressing subpopulation of growth hormone secretagogue receptor cells are distinct from kisspeptin,tyrosine hydroxylase,and RFamide-related peptide neurons in mice

Ghrelin acts on the growth hormone secretagogue receptor (GHSR) in the brain to elicit changes in physiological functions. It is associated with the neural control of appetite and metabolism, however central ghrelin also affects fertility. Central ghrelin injection in rats suppresses luteinizing hormone (LH) concentrations and pulse frequency. Although ghrelin suppresses LH and regulates kisspeptin mRNA in the anteroventral periventricular/periventricular nucleus (AVPV/PeN), there is no neuroanatomical evidence linking GHSR neural circuits to kisspeptin neurons. In this study, we first determined coexpression of GHSR and GnRH neurons using a GHSR-eGFP reporter mouse line. Using dual-label immunohistochemistry, we saw no coexpression. GHSR-eGFP expressing cells were present in the AVPV/PeN and over 90% of these expressed estrogen receptor-α (ERα). Despite this, we observed no evidence of GHSR-eGFP/kisspeptin coexpressing neurons in the AVPV/PeN. To further examine the phenotype of GHSR-eGFP cells in the AVPV/PeN, we determined coexpression with tyrosine hydroxylase (TH) and showed virtually no coexpression in the AVPV/PeN (<2%). We also observed no coexpression of GHSR-eGFP and RFamide-related peptide-3 (RFRP3) neurons in the dorsomedial hypothalamic nucleus. Importantly, we observed that approximately half of the GHSR-eGFP cells in the AVPV coexpressed Ghsr mRNA (as determined by in situ hybridization) so these data should be interpreted accordingly. Although ghrelin influences the hypothalamic reproductive axis, our data using a GHSR-eGFP reporter suggests ghrelin regulates neurons expressing ERα but does not directly act on GnRH, kisspeptin, TH, or RFRP3 neurons, as little or no GHSR-eGFP coexpression was observed.     

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.