Peripheral ghrelin interacts with orexin neurons in glucostatic signalling

Ghrelin interactions with glycemia in appetite control as well as the potential mechanisms involving the orexin and melanin-concentrating hormone (MCH) neurons in the orexigenic ghrelin signals were investigated by using a specific anti-ghrelin antibody (AGA). Our results confirm that peripheral ghrelin is an important signal in meal initiation and appetite. Employing immunohistochemistry techniques, we found that c-fos positive neurons in the lateral hypothalamus (LH) and perifornical area (PFA) increased after insulin or 2-deoxyglucose administration. Moreover, we have also demonstrated that peripheral ghrelin blockade by the AGA, reduces the orexigenic signal induced by insulin and 2-DG administration probably partly producing a decrease of c-fos immunoreactivity in the LH and PFA as well as a lower activation of orexin neurons. In contrast, the c-fos positive MCH neurons were not apparently affected. In summary, our findings suggest that peripheral ghrelin plays an important role in regulatory "glucostatic" feeding mechanisms by means of its role as a "hunger" signal affecting the LH and PFA areas, which may contribute to energy homeostasis through orexin neurons.     

Dissociation of hypothalamic noradrenergic activity and sympathoadrenal responses to recurrent hypoglycemia

This study evaluated whether attenuation of sympathoadrenal responses to recurrent hypoglycemia is mediated by diminished noradrenergic activity in the hypothalamus. Male Sprague-Dawley rats received either once daily insulin (1.0 units/kg) injections or an equal administration of saline for 3 days. Both groups received an administration of insulin on the fourth day, during which blood glucose and plasma catecholamines were determined, and extracellular norepinephrine (NE) in the ventromedial hypothalamus (VMH) or paraventricular hypothalamic nucleus (PVN) was monitored with microdialysis. The peak response of plasma epinephrine to insulin-induced hypoglycemia (nadir approximately 3.2 mmol/l) was significantly reduced during the fourth hypoglycemic episode (774 +/- 134 pg/ml) compared with the first episode (2,561 +/- 410 pg/ml, P < 0.001). Baseline levels of extracellular NE were elevated approximately 25% (P = 0.07) in the VMH and approximately 46% (P = 0.03) in the PVN after multiple hypoglycemic episodes. There was no difference in noradrenergic activity during the first or fourth hypoglycemic episode in either brain area. The reduced sympathoadrenal output after recurrent hypoglycemia is likely postsynaptic from hypothalamic NE release or is mediated via a collateral pathway.     

DISTRIBUTION OF GAMMA-AMINOBUTYRIC ACID (GABA) IN THE RAT HYPOTHALAMUS: FUNCTIONAL CORRELATES OF GABA WITH ACTIVITIES OF APPETITE CONTROLLING MECHANISMS

—In the hypothalamus, the highest GABA content (approx. 26 nmol/mm³) was constantly observed in the lateral hypothalamic area (LHA). In other parts of the hypothalamus uneven distribution of GABA was also observed, but areas showing high concentration of GABA did not coincide with the locations of various hypothalamic nuclei. In the LHA, which is known to contain a feeding centre, the anterior part (6.4 and 6.0 mm anterior (A 6.4 and A 6.0) respectively to the vertical zero plane of de Groot) showed a remarkably high content of GABA. The GABA content in the LHA at A 6.4 was decreased during the initial phase of insulin hypoglycemia and, in contrast, showed a significant increase following hyperglycemia induced by alloxan administration. In the ventromedial nucleus (VMH) of the hypothalamus, which is known to contain a satiety centre, the GABA content was increased during the initial phase of insulin hypoglycemia. The results suggest that both certain parts of the LHA and VMH contain or receive GABA-inhibitory neurons and that these neurons may play important physiological roles in controlling functional states of the feeding and satiety centres in the hypothalamus.     

Cell activation in the hypothalamus after exposure to an antigen (based on c-fos gene expression)

Increase of the c-fos mRNA positive cells number was insignificant in 30 min. following activation of the rat hypothalamic structures with the tetanus toxoid (TT). Elevation of the c-fos mRNA positive cells number occurred in the hypothalamic' posterior (PHA), lateral (LHA), anterior (AHA), areas dorsomedial (DMH), and ventromedial (VMH) nuclei within 2 hours of the TT administration. In 6 hours the c-fos mRNA positive cells number decreased in PHA, LHA, DMH. The c-fos mRNA expression was stable in arquate and supraoptic hypothalamic nuclei following either the TT or saline administration.     

Oxytocinergic circuit from paraventricular and supraoptic nuclei to arcuate POMC neurons in hypothalamus

Intracerebroventricular injection of oxytocin (Oxt), a neuropeptide produced in hypothalamic paraventricular (PVN) and supraoptic nuclei (SON), melanocortin-dependently suppresses feeding. However, the underlying neuronal pathway is unclear. This study aimed to determine whether Oxt regulates propiomelanocortin (POMC) neurons in the arcuate nucleus (ARC) of the hypothalamus. Intra-ARC injection of Oxt decreased food intake. Oxt increased cytosolic Ca²⁺ in POMC neurons isolated from ARC. ARC POMC neurons expressed Oxt receptors and were contacted by Oxt terminals. Retrograde tracer study revealed the projection of PVN and SON Oxt neurons to ARC. These results demonstrate the novel oxytocinergic signaling from PVN/SON to ARC POMC, possibly regulating feeding.     

PYY(3-36) induces Fos in the arcuate nucleus and in both catecholaminergic and non-catecholaminergic neurons in the nucleus tractus solitarius of rats

Peptide YY (3-36) [PYY(3-36)] inhibits feeding in rodents, nonhuman primates and humans, yet the neural circuits underlying this action remain to be determined. Here we assessed whether PYY(3-36) inhibits feeding by activating neurons in forebrain and hindbrain sites containing Y2 receptors and linked to control of food intake, or in hindbrain sites immediately downstream of vagal afferent neurons. Rats received an anorexigenic dose of PYY(3-36), and the number of neurons expressing Fos, an indicator of neuronal activation, was determined in anterior hypothalamus (AH), arcuate nucleus (ARC), dorsomedial hypothalamus (DMH), lateral hypothalamus (LH), ventromedial hypothalamus (VMH), central nucleus of the amygdala (CeA), area postrema (AP), and caudal medial nucleus tractus solitarius (cmNTS), commissural NTS (cNTS), and gelatinosus NTS (gNTS). Expression of tyrosine hydroxylase (TH), an indicator of catecholamine synthesis, was also measured in the cmNTS. PYY(3-36) increased Fos in ARC, cmNTS, gNTS and AP. Approximately 10% of Fos+ neurons in the cmNTS were TH+. These results suggest that PYY(3-36) inhibits feeding through direct activation of ARC neurons, and direct and/or indirect activation via vagal afferent nerves of cmNTS, gNTS and AP, including some catecholaminergic neurons in the cmNTS.     

Hypothalamic administration of cAMP agonist/PKA activator inhibits both schedule feeding and NPY-induced feeding in rats

Following central administration, neuropeptides that decrease the level of cAMP induce feeding. Conversely, cAMP activating neuropeptides tend to elicit satiety. When the inhibitory effect of neuropeptide Y (NPY) on the hypothalamic cAMP production was blocked by pertussis toxin, the potent orexigenic effect of NPY was lost. These findings suggest that there may be a link between hypothalamic cAMP and the central regulation of food intake. In this report, we show that the injection of the membrane-permeable cAMP agonist, adenosine-3',5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP), into perifornical hypothalamus (PFH) significantly inhibited schedule-induced and NPY-induced food intake for up to 4h. This inhibitory effect was normalized within 24h. A taste aversion could not be conditioned to Sp-cAMP treatment, suggesting that the anorectic response was not due to malaise. Sp-cAMP administration significantly increased the active protein kinase A (PKA) activity in dorsomedial (DMH) and ventromedial (VMH), but not in lateral (LH) hypothalamus. Consistently, food deprivation lowered, while refeeding normalized endogenous cAMP content in DMH and VMH, but not in LH areas. No significant effect of adenosine-3',5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP, cAMP antagonist) was observed on hypothalamic PKA activity, schedule-induced, or NPY-induced food intake. These findings suggest that the increase in cAMP level and PKA activity in DMH and VMH areas may trigger a satiety signal.     

Ghrelin-containing neuron in cerebral cortex and hypothalamus linked with the DVC of brainstem in rat

Ghrelin is a newly discovered brain-gut peptide and an endogenous ligand for growth hormone secretagogues receptor (GHS-R). Ghrelin and GHS-R present extensively in central and peripheral tissues such as stomach, brain and other organs of rodent and human, which suggest it has multiple biological effects. It has been reported that ghrelin has significant role in the regulation of energy homeostasis, food intake and appetite. The organization of central circuitry appears to play an important role in integrating orexigenic effects of ghrelin, but the detail is not fully clear. In this study, we examined the expression of ghrelin, ghrelin mRNA and GHS-R mRNA in cerebrum and brainstem by RT-PCR and immunofluorescence histochemistry, and analyzed the connection among the cerebral cortex, hypothalamus, dorsal vagal complex (DVC). The results showed that the positive staining of ghrelin was found on the pyramidal neuron of layer V in the sensorimotor area of cerebral cortex, cingulate gyrus, as well as in the neuron of lateral hypothalamus (LH), PVN and ARC. The expression of ghrelin mRNA and GHS-R mRNA were also found in the sensorimotor cortex and hypothalamus by method of RT-PCR. The GHS-R mRNA was also found in the DVC of medulla oblongata. Other finding is that the FG/ghrelin dual labeled neurons were found in LH of hypothalamus (not in cortex). The ghrelin-containing neuron in the LH projects its axon to the DVC with the method of retrograde tracing. In conclusion, the ghrelin neurons are located not only in hypothalamus (LH, PVN, ARC), but also in the cortex (sensorimotor area, cingular gyrus), and the fibers of ghrelin neurons in hypothalamus projected directly to the DVC. It suggests that ghrelin plays its role from hypothalamus to brainstem as a neurotransmitter or neuromodulator to regulate function of vagal nuclei in brainstem.     

Hypothalamic site of action for N-methyl-D-aspartate (NMDA) on LH secretion

Excitatory amino acids have been shown to increase luteinizing hormone (LH) secretion following ventricular or systemic administration. In the present study we attempted to determine possible hypothalamic sites of action for the potent excitatory amino acid agonist, N-methyl-D-aspartate (NMDA). The ability of NMDA to enhance LH release was tested in male rats following infusion into the medial preoptic nucleus (MPO), anterior hypothalamic nucleus (AHY), ventromedial hypothalamic nucleus (VMH), and arcuate nucleus (ARC). In the MPO, infusion of 50 or 500 pmole NMDA increased pituitary LH secretion, resulting in a 2-7 fold increase in plasma LH. The 50 pmole dose was selected to test more caudal hypothalamic sites. Plasma LH levels were not affected following microinfusion of NMDA (50 pmole) into the AHY, VMH, and ARC. The present results indicate a regional specificity for NMDA in the enhancement of LH secretion. This regional specificity may reflect either a greater density of LHRH perikarya in the MPO or the presence of specific amino acid receptors on neuronal elements in the MPO, but not on neuronal elements in the other areas tested.     

Effects of ghrelin on neuronal activity in the ventromedial nucleus of the hypothalamus in infantile rats: An in vitro study

Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue (GHS) receptor (GHS-R) and a potent stimulant for GH secretion even in infantile rats before puberty. Although the ventromedial nucleus of the hypothalamus (VMH) might be a site of action for ghrelin to induce GH release, the electrophysiological effect of ghrelin on VMH neurons in infantile rats remains to be elucidated. Thus, the purpose of the present study was to investigate the effect of ghrelin on VMH neurons using hypothalamic slices of infantile rats. Ghrelin excited a majority of VMH neurons in a concentration-dependent manner. VMH neurons that were excited by GH releasing peptide-6 (GHRP-6), a synthetic GHS, were also excited by ghrelin and vice versa. Repeated application of ghrelin to the same VMH neuron decreased progressively the excitatory responses depending on the number of times it was administered. The excitatory effect of ghrelin on VMH neurons in normal artificial cerebrospinal fluid (ACSF) persisted in low Ca²⁺-high Mg²⁺ ACSF. The present results indicate that (1) ghrelin excites a majority of VMH neurons dose-dependently and postsynaptically and (2) the excitatory effects of ghrelin are mimicked by GHRP-6 and desensitized by repeated applications of ghrelin.     

Activation of 5-hydroxytryptamine-1A receptors suppresses cardiovascular responses evoked from the paraventricular nucleus

Activation of central 5-hydroxytryptamine-1A (5-HT(1A)) receptors powerfully inhibits stress-evoked cardiovascular responses mediated by the dorsomedial hypothalamus (DMH), as well as responses evoked by direct activation of neurons within the DMH. The hypothalamic paraventricular nucleus (PVN) also has a crucial role in cardiovascular regulation and is believed to regulate heart rate and renal sympathetic activity via pathways that are independent of the DMH. In this study, we determined whether cardiovascular responses evoked from the PVN are also modulated by activation of central 5-HT(1A) receptors. In anesthetized rats, the increases in heart rate and renal sympathetic nerve activity evoked by bicuculline injection into the PVN were greatly reduced (by 54% and 61%, respectively) by intravenous administration of (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), an agonist of 5-HT(1A) receptors, but were then completely restored by subsequent administration of WAY-100635, a selective antagonist of 5-HT(1A) receptors. Microinjection of 8-OH-DPAT directly into the PVN did not significantly affect the responses to bicuculline injection into the PVN, nor did systemic administration of WAY-100635 alone. In control experiments, a large renal sympathoexcitatory response was evoked from both the PVN and DMH but not from the intermediate region in between; thus the evoked responses from the PVN were not due to activation of neurons in the DMH. The results indicate that activation of central 5-HT(1A) receptors located outside the PVN powerfully inhibits the tachycardia and renal sympathoexcitation evoked by stimulation of neurons in the PVN.     

Hypothalamic adrenergic receptor changes in the metabolic syndrome of genetically obese (ob/ob) mice

The genetically, seasonally, and diet-induced obese, glucose-intolerant states in rodents, including ob/ob mice, have each been associated with elevated hypothalamic levels of norepinephrine (NE). With the use of quantitative autoradiography on brain slices of 6-wk-old obese (ob/ob) and lean mice, the adrenergic receptor populations in several hypothalamic nuclei were examined. The binding of [(125)I]iodocyanopindolol to beta(1)- and beta(2)-adrenergic receptors in ob/ob mice was significantly increased in the paraventricular hypothalamic nucleus (PVN) by 30 and 38%, in the ventromedial hypothalamus (VMH) by 23 and 72%, and in the lateral hypothalamus (LH) by 10 and 15%, respectively, relative to lean controls. The binding of [(125)I]iodo-4-hydroxyphenyl-ethyl-aminomethyl-tetralone to alpha(1)-adrenergic receptors was also significantly increased in the PVN (26%), VMH (67%), and LH (21%) of ob/ob mice. In contrast, the binding of [(125)I]paraiodoclonidine to alpha(2)-adrenergic receptors in ob/ob mice was significantly decreased in the VMH (38%) and the dorsomedial hypothalamus (17%) relative to lean controls. This decrease was evident in the alpha(2A)- but not the alpha(2BC)-receptor subtype. Scatchard analysis confirmed this decreased density of alpha(2)-receptors in ob/ob mice. Together with earlier studies, these changes in hypothalamic adrenergic receptors support a role for increased hypothalamic NE activity in the development of the metabolic syndrome of ob/ob mice.     

Prior hypoglycemia enhances glucose responsiveness in some ventromedial hypothalamic glucosensing neurons

Antecedent insulin-induced hypoglycemia (IIH) reduces adrenomedullary responses (AMR) to subsequent bouts of hypoglycemia. The ventromedial hypothalamus [VMH: arcuate (ARC) + ventromedial nuclei] contains glucosensing neurons, which are thought to be mediators of these AMR. Since type 1 diabetes mellitus often begins in childhood, we used juvenile (4- to 5-wk-old) rats to demonstrate that a single bout of IIH (5 U/kg sc) reduced plasma glucose by 24% and peak epinephrine by 59% 1 day later. This dampened AMR was associated with 46% higher mRNA for VMH glucokinase, a key mediator of neuronal glucosensing. Compared with neurons from saline-injected rats, ventromedial nucleus glucose-excited neurons from insulin-injected rats demonstrated a leftward shift in their glucose responsiveness (EC50 = 0.45 and 0.10 mmol/l for saline and insulin, respectively, P = 0.05) and a 31% higher maximal activation by glucose (P = 0.05), although this maximum occurred at a higher glucose concentration (saline, 0.7 vs. insulin, 1.5 mmol/l). Although EC50 values did not differ, ARC glucose-excited neurons had 19% higher maximal activation, which occurred at a lower glucose concentration in insulin- than saline-injected rats (saline, 2.5 vs. insulin, 1.5 mmol/l). In addition, ARC glucose-inhibited neurons from insulin-injected rats were maximally inhibited at a fivefold lower glucose concentration (saline, 2.5 vs. insulin, 0.5 mmol/l), although this inhibition declined at >0.5 mmol/l glucose. These data suggest that the increased VMH glucokinase after IIH may contribute to the increased responsiveness of VMH glucosensing neurons to glucose and the associated blunting of the AMR.     

PVN activation is suppressed by repeated hypoglycemia but not antecedent corticosterone in the rat

The mechanism(s) underlying hypoglycemia-associated autonomic failure (HAAF) are unknown. To test the hypothesis that the activation of brain regions involved in the counterregulatory response to hypoglycemia is blunted with HAAF, rats were studied in a 2-day protocol. Neuroendocrine responses and brain activation (c-Fos immunoreactivity) were measured during day 2 insulin-induced hypoglycemia (0.5 U insulin x 100 g body x wt(-1) x h(-1) iv for 2 h) after day 1 hypoglycemia (Hypo-Hypo) or vehicle. Hypo-Hypo animals demonstrated HAAF with blunted epinephrine, glucagon, and corticosterone (Cort) responses and decreased activation of the medial hypothalamus [the paraventricular (PVN), dorsomedial (DMH), and arcuate (Arc) nuclei]. To evaluate whether increases in day 1 Cort were responsible for the decreased hypothalamic activation, Cort was infused intracerebroventricularly (72 microg) on day 1 and the response to day 2 hypoglycemia was measured. Intracerebroventricular Cort infusion failed to alter the neuroendocrine response to day 2 hypoglycemia, despite elevating both central nervous system and peripheral Cort levels. However, day 1 Cort blunted responses in two of the same hypothalamic regions as Hypo-Hypo (the DMH and Arc) but not in the PVN. These results suggest that decreased activation of the PVN may be important in the development of HAAF and that antecedent exposure to elevated levels of Cort is not always sufficient to produce HAAF.     

Ghrelin indirectly activates hypophysiotropic CRF neurons in rodents

Ghrelin is a stomach-derived hormone that regulates food intake and neuroendocrine function by acting on its receptor, GHSR (Growth Hormone Secretagogue Receptor). Recent evidence indicates that a key function of ghrelin is to signal stress to the brain. It has been suggested that one of the potential stress-related ghrelin targets is the CRF (Corticotropin-Releasing Factor)-producing neurons of the hypothalamic paraventricular nucleus, which secrete the CRF neuropeptide into the median eminence and activate the hypothalamic-pituitary-adrenal axis. However, the neural circuits that mediate the ghrelin-induced activation of this neuroendocrine axis are mostly uncharacterized. In the current study, we characterized in vivo the mechanism by which ghrelin activates the hypophysiotropic CRF neurons in mice. We found that peripheral or intra-cerebro-ventricular administration of ghrelin strongly activates c-fos--a marker of cellular activation--in CRF-producing neurons. Also, ghrelin activates CRF gene expression in the paraventricular nucleus of the hypothalamus and the hypothalamic-pituitary-adrenal axis at peripheral level. Ghrelin administration directly into the paraventricular nucleus of the hypothalamus also induces c-fos within the CRF-producing neurons and the hypothalamic-pituitary-adrenal axis, without any significant effect on the food intake. Interestingly, dual-label immunohistochemical analysis and ghrelin binding studies failed to show GHSR expression in CRF neurons. Thus, we conclude that ghrelin activates hypophysiotropic CRF neurons, albeit indirectly.     

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.     

A mechanism by which primary or secondary hypothalamic involvement results in the development of insulin-dependent diabetes mellitus (IDDM)

A literature survey and hypothesis is presented in which it is concluded that an intracellular ventromedial hypothalamic (VMH) glucopenia results in a bibrachial response consisting of adenohypophysial release of growth hormone and ACTH as well as sympathetic discharge, both of which act to elevate plasma glucose and remove the VMH glucopenia. This glucopenia may occur as a result of either a deficiency of circulating insulin or alterations in the kinetic properties of the VMH cellular insulin receptor. Two mechanisms for the development of insulin dependent diabetes mellitus (IDDM) are presented: (1) a defect in VMH glucose transport and/or metabolism such that a VMH glucopenia occurs with a subsequent bibrachial response. The result of this is glucose overproduction and a chronic excess glucose stimulus will eventually cause B-cell exhaustion (primary hypothalamic involvement). (2) reduction of the B-cell population by chemical, genetic and/or viral interactions with a consequential insulopenia results in a VMH glucopenia (secondary to a reduced glucose transport into the VMH cells) and causes a bibrachial response. This VMH response may temporarily restore plasma glucose balance but a chronically enhanced counter-regulatory response to maintain this balance will eventually stress the remaining B-cell population and cause further reductions in B-cell numbers (secondary hypothalmic involvement).     

Neural basis of orexigenic effects of ghrelin acting within lateral hypothalamus

Ghrelin stimulates feeding when administered centrally and peripherally. The lateral hypothalamus (LH) is thought to mediate ghrelin-induced hyperphagia. Thus, we examined central mechanisms underlying feeding generated by LH ghrelin. We determined that 0.3nmol of LH-injected ghrelin was the lowest dose increasing food consumption and it induced Fos immunoreactivity (IR; a marker of neuronal activation) in feeding-related brain areas, including the hypothalamic paraventricular, arcuate, and dorsomedial nuclei, amygdala, and nucleus of the solitary tract. Also, LH ghrelin induced Fos IR in LH orexin neurons. We conclude that the LH, as part of larger central circuitry, integrates orexigenic properties of ghrelin.     

c-Fos expression in hypothalamic nuclei of food-entrained rats

The present study aimed to identify the hypothalamic nuclei involved with food entrainment by using c-Fos-like immunoreactivity (c-Fos-IR) as a marker of functional activation. We studied rats entrained 3 wk to restricted feeding schedules (RF), their ad libitum (AL) controls, and the persistence of c-Fos-IR temporal patterns in entrained-fasted rats. In addition, we included 22-h fasting and 22-h fasting-refeeding groups as controls of fasting and refeeding acute effects. Diurnal patterns of c-Fos-IR were observed in the tuberomammilar nucleus (TM) and suprachiasmatic nucleus (SCN) in AL rats. In all nuclei, except the SCN and ventromedial nucleus (VMH), restricted feeding schedules imposed a temporal pattern of increased c-Fos-IR around mealtime. An increase in c-Fos-IR before and after meal time was observed in dorsomedial nucleus (DMH), lateral nucleus (LH), perifornical area (PeF), and TM, and a marked increase was observed in the paraventricular nucleus (PVN) after feeding. Food-entrained c-Fos-IR patterns persisted after 3 days in fasting in DMH, LH, and PeF. Present data suggest that FEO might not rely on a single nucleus and rather may be a distributed system constituted of interacting nuclei in which the PVN is mainly involved with the response to signals elicited by food ingestion and, therefore, with the entraining pathway. We can suggest that the PeF and TM may be involved with the arousal state during food anticipation and the DMH and LH with the time-keeping mechanism of FEO or its output.