Central and peripheral alytesin cause short-term anorexigenic effects in neonatal chicks

We studied the effects of alytesin, a natural analogue of bombesin, on appetite-related responses and behaviors in neonatal chicks. Chicks responded to both intracerebroventricular (ICV) and peripheral injections of alytesin with short-term reduced feed intake. ICV alytesin caused reduced short-term water intake when feed was present, but we determined this effect was secondary to feed intake since an effect on water intake was not detected in feed-restricted alytesin-treated chicks. The anorexigenic effect of both ICV and peripheral alytesin may be mediated at the hypothalamus, since all hypothalamic nuclei studied; regio lateralis hypothalami, nucleus dorsomedialis hypothalami, nucleus paraventricularis magnocellularis, nucleus perventricularis hypothalami, nucleus infundibuli hypothalami and the nucleus ventromedialis hypothalami, were activated as evident by increased c-Fos immunoreactivity. Central alytesin did not cause increased behaviors that were unrelated to ingestion and did not cause anxiety-related behavior patterns. Additionally, central alytesin did not affect pecking efficacy. We conclude that both ICV and peripheral alytesin injections induce anorexigenic effects in chicks, and the hypothalamus is involved. While the anorexigenic effects of alytesin and bombesin appear to be conserved across species, the two peptides may differ in other behavioral responses and central mechanisms of action.     

Hypothalamic mechanisms associated with corticotropin-releasing factor-induced anorexia in chicks

Central administration of corticotropin-releasing factor (CRF), a 41-amino acid peptide, is associated with potent anorexigenic effects in rodents and chickens. However, the mechanism underlying this effect remains unclear. Hence, the objective of the current study was to elucidate the hypothalamic mechanisms that mediate CRF-induced anorexia in 4 day-old Cobb-500 chicks. After intracerebroventricular (ICV) injection of 0.02 nmol of CRF, CRF-injected chicks ate less than vehicle chicks while no effect on water intake was observed at 30 min post-injection. In subsequent experiments, the hypothalamus samples were processed at 60 min post-injection. The CRF-injected chicks had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), dorsomedial nucleus (DMN), ventromedial hypothalamus (VMH), and paraventricular nucleus (PVN) of the hypothalamus than vehicle-treated chicks. CRF injection was associated with decreased whole hypothalamic mRNA abundance of neuropeptide Y receptor sub-type 1 (NPYR1). In the ARC, CRF-injected chicks expressed more CRF and CRF receptor sub-type 2 (CRFR2) mRNA but less agouti-related peptide (AgRP), NPY, and NPYR1 mRNA than vehicle-injected chicks. CRF-treated chicks expressed greater amounts of CRFR2 and mesotocin mRNA than vehicle chicks in the PVN and VMH, respectively. In the DMN, CRF injection was associated with reduced NPYR1 mRNA. In conclusion, the results provide insights into understanding CRF-induced hypothalamic actions and suggest that the anorexigenic effect of CRF involves increased CRFR2-mediated signaling in the ARC and PVN that overrides the effects of NPY and other orexigenic factors.     

Substance P is associated with hypothalamic paraventricular nucleus activation that coincides with increased urotensin 2 mRNA in chicks

Exogenous administration of substance P (SP) exerts anorexigenic effects in both chicks and rats, but the central mechanism mediating this response is poorly understood. Therefore, this study was designed to elucidate mechanisms of SP-induced anorexia using chicks as models. Chicks that received intracerebroventricular (ICV) injections of SP dose-dependably reduced their food intake with no effect on water intake. Next, the diencephalon was isolated from SP-injected chicks and mRNA expression of neuropeptide Y (NPY), corticotropin releasing factor (CRF), urocortin 3 (UCN 3) and CRF receptors were measured but were not affected. When measured in the hypothalamus, mRNA abundance of these and NPY receptors, urotensin 2 (UTS2) and melanocortin receptor 4 (MCR4) were not affected by SP-injection. Quantification of c-Fos immunoreactivity in appetite-associated hypothalamic nuclei demonstrated that the paraventricular nucleus (PVN) was activated in SP-injected chicks. Finally, in the PVN isolated from SP-injected chicks, there was increased expression of UTS2 mRNA while CRF and UCN3 were not affected. Thus, the anorexigenic effects of SP appear to be mediated by PVN activation and may involve UTS2.     

The central anorexigenic mechanism of adrenocorticotropic hormone involves the caudal hypothalamus in chicks

Adrenocorticotropic hormone (ACTH), consisting of 39 amino acids, is most well-known for its involvement in an organism's response to stress. It also participates in satiety, as exogenous ACTH causes decreased food intake in rats. However, its anorexigenic mechanism is not well understood in any species and its effect on appetite is not reported in the avian class. Thus, the present study was designed to evaluate central ACTH's effect on food intake and to elucidate the mechanism mediating this response using broiler chicks. Chicks that received intracerebroventricular (ICV) injection of 1, 2, or 4 nmol of ACTH reduced food intake, under both ad libitum and 180 min fasted conditions. Water intake was also reduced in ACTH-injected chicks under both feeding conditions, but when measured without access to feed it was not affected. Blood glucose was not affected in either feeding condition. Following ACTH injection, c-Fos immunoreactivity was quantified in key appetite-associated hypothalamic nuclei including the ventromedial hypothalamus (VMH), dorsomedial hypothalamus, lateral hypothalamus (LH), arcuate nucleus (ARC) and the parvo- and magno-cellular portions of the paraventricular nucleus. ACTH-injected chicks had increased c-Fos immunoreactivity in the VMH, LH, and ARC. Hypothalamus was collected at 1 h post-injection, and real-time PCR performed to measure mRNA abundance of some appetite-associated factors. Neuropeptide Y, pro-opiomelanocortin, glutamate decarboxylase 1, melanocortin receptors 2–5, and urocortin 3 mRNA abundance was not affected by ACTH treatment. However, expression of corticotropin releasing factor (CRF), urotensin 2 (UT), agouti-related peptide (AgRP), and orexin (ORX), and melanocortin receptor 1 (MC1R) mRNA decreased in the hypothalamus of ACTH-injected chicks. In conclusion, ICV ACTH causes decreased food intake in chicks, and is associated with VMH, LH, and ARC activation, and a decrease in hypothalamic mRNA abundance of CRF, UT, AgRP, ORX and MC1R.     

Anorexigenic effects of substance P in Coturnix japonica

Substance P (SP) is an 11-amino acid tachykinin-related peptide that has anorexigenic effects in birds and mammals although the central mechanism is not well understood. Hence, the objective was to identify appetite-associated hypothalamic mechanisms in Japanese quail (Coturnix japonica). Seven days post-hatch, quail were intracerebroventricularly injected with 0, 0.25, 0.5 or 1.0 nmol of SP and monitored for 180 min. On a cumulative basis, quail that received 0.5 and 1.0 nmol of SP consumed less food for 90 min post-injection. On a non-cumulative basis, food intake was reduced in 0.5 nmol-injected birds at 30 min post-injection. Water intake was not affected. A comprehensive behavior analysis was performed, revealing that SP-injected chicks displayed less feeding pecks and reduced locomotion compared to vehicle-injected birds. To identify molecular mechanisms, the hypothalamus was isolated at 1 h post-injection and real-time PCR was performed to measure mRNA. Agouti-related peptide (AgRP) mRNA was reduced in SP-injected chicks. Immunohistochemistry was used to quantify c-Fos-expressing cells in appetite-associated hypothalamic nuclei. There were more reactive cells in the lateral hypothalamus (LH) and the paraventricular nucleus (PVN) of SP- than vehicle-injected chicks. The LH and PVN were collected for gene expression analysis. Corticotropin-releasing factor (CRF) and urotensin 2 (UTS2) mRNAs were greater in SP- than vehicle-injected chicks in the PVN. In the LH, CRF receptor sub-type 2 (CRFR2) mRNA was greater and kappa opioid receptor mRNA was reduced in SP- compared to vehicle-injected quail. Thus, SP induces a potent anorexia in quail that coincides with increased LH-specific CRFR2 mRNA and increased UTS2 mRNA in the PVN. Future studies will evaluate whether SP-induced anorexigenic effects are mediated through CRF receptors.     

Food intake elicited by central administration of orexins/hypocretins: identification of hypothalamic sites of action

Orexin A and B, a recently identified pair of neuropeptides, are produced in perikarya located in the lateral and perifornical hypothalamus (LH and PFH). Immunoreactive fibers from these neurons innervate several nuclei in the hypothalamus. Orexin A and orexin B stimulate feeding when administered intracerebroventricularly to rats. To identify the specific sites of orexin action, orexin A and B were microinjected into a number of hypothalamic and extrahypothalamic sites in rats. Orexin A was found to enhance food intake when injected into four hypothalamic sites, the paraventricular nucleus (PVN), the dorsomedial nucleus (DMN), LH and the perifornical area, but was ineffective in the arcuate nucleus (ARC), the ventromedial nucleus (VMN), and the preoptic area (POA) as well as the central nucleus of the amygdala (CeA) and nucleus of the tractus solitarius (NTS). Orexin B was not effective at any site tested. These findings demonstrate that orexin A receptive sites for stimulation of food intake exist primarily in a narrow band of neural tissue within the hypothalamus that is known to be involved in control of energy homeostasis.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

Central visfatin causes orexigenic effects in chicks

Intracerebroventricular injection of visfatin caused increased feed intake and pecking efficiency, but did not affect water intake in chicks. Visfatin-treated chicks had increased c-Fos immunoreactivity in the lateral hypothalamus, decreased reactivity in the ventromedial hypothalamus and the dorsomedial hypothalamus, infundibular nucleus, periventricular nucleus, paraventricular nucleus were not affected. A low dose of visfatin increased locomotion. We conclude that intracerebroventricular injection of visfatin causes orexigenic effects in chicks.     

Microinjection of RFRP-1 in the central nucleus of amygdala decreases food intake in the rat

Several members of the RFamide peptide family are known to have role in the regulation of feeding. For example, neuropeptide FF and prolactin-releasing peptide cause anorexigenic, while 26RFa and QRFP result in orexigenic effects in rodents. I.c.v. microinjection of neuropeptide RFRP-1 significantly reduced food and water intake in chicks. However, feeding related effects of RFRP-1 have not been studied in mammals yet. The central part of amygdala (CeA) is essentially involved in the regulation of feeding and body weight. RFRP-1 positive nerve cells were detected in the rat hypothalamus and RFRP-1 immunoreactive fibers were identified in the CeA. RFRP analogs bind with relatively high affinity to the NPFF1 and NPFF2 receptors (NPFF-R). RFRP-1 has potent activity for NPFF1. Significant expression of NPFF1 was detected in the CeA. To evaluate the role of RFRP-1 in feeding regulation rats were microinjected with different doses of RFRP-1 and their food intake were quantified over a 60min period. Liquid food intake of male Wistar rats was measured after bilateral intraamygdaloid administration of RFRP-1 (25, 50 or 100ng/side, RFRP-1 dissolved in 0.15M sterile NaCl/0.4μl, respectively). The 50ng dose of RFRP-1 microinjections resulted in significant decrease of food intake. The 25 and 100ng had no effect. Action of 50ng (37.8pmol) RFRP-1 was eliminated by 20ng (41.4pmol) RF9 NPFF-R antagonist pretreatment. In open-field test 50ng RFRP-1 did not modify spontaneous locomotor activity and general behavior of animals did not change. Our results are the first reporting that RFRP-1 injected to the CeA result in a decrease of liquid food consumption. This is a receptor-linked effect because it was eliminated by a NPFF-R selective antagonist.     

Xenin reduces feed intake by activating the ventromedial hypothalamus and influences gastrointestinal transit rate in chicks

This study was conducted to determine the effects of xenin on appetite related processes in chicks. Chicks were centrally and peripherally administered xenin, and feed and water intake were quantified. Chicks responded with a linear dose-dependent decrease in feed intake to central xenin and had a quadratic type response to peripheral administration. Water intake was not affected by treatment. To determine if the lateral hypothalamus (LH) or ventromedial hypothalamus (VMH) was involved in this effect, chicks were both centrally and peripherally injected with xenin and an immunocytochemistry assay for c-Fos was conducted. Central and peripheral xenin caused increased activation of the VMH but had no effect on the LH. Finally, to determine if gastrointestinal transit rate was affected, chicks received central xenin and were gavaged with chicken feed slurry containing a visible marker. Chicks exhibited a quadratic dose-dependent response to transit rate after central xenin. These results suggest that xenin affects feeding and gastrointestinal motility through hypothalamic interactions in chicks.     

Effects of PVN lesions on the responsiveness of female rats to estradiol.

Previous research has shown that the paraventricular nucleus of the hypothalamus (PVN) is an important site of action for the effects of estradiol on feeding behavior. The recent finding that estrogenic stimulation of the PVN lowers food intake without inducing lordosis suggests that the effects of estradiol on feeding and sexual behaviors are organized separately within the brain. Whether the effects of estradiol on food intake can be attenuated by PVN lesions is therefore a question of practical and theoretical interest. In this experiment we examined the behavioral responsiveness of females with PVN lesions to peripheral treatment with estradiol. 32 adult, female rats received either bilateral or sham lesions of the PVN. All subjects were ovariectomized 2 weeks after the lesion. 2 Weeks following ovariectomy, half of the animals were injected with 2 micrograms of estradiol benzoate (EB) for 3 days, and half were injected with the oil vehicle. 10 days later, the treatment conditions for each subject (oil or EB) were reversed. Histological analysis indicated that 9 females had bilateral lesions of the PVN and 4 had bilateral lesions of the dorsomedial nucleus of the hypothalamus (DMN); 11 animals received sham lesions. Compared with oil treatment, EB injections significantly lowered water intake and body weight gain in all groups. However, food intake was suppressed in the DMN and sham but not in PVN-lesioned females. In addition, statistical analyses indicated that EB treatment induced similar levels of female sexual behavior in all groups. Thus, PVN lesions did not interfere with the ability of estradiol to stimulate lordosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Central administration of neuropeptide FF causes activation of oxytocin paraventricular hypothalamic neurones that project to the brainstem

Neuropeptide FF (NPFF), a morphine modulatory peptide, is emerging as an important neuromodulator in the context of central autonomic and neuroendocrine regulation. NPFF immunoreactivity and receptors have been identified in discrete autonomic regions within the brain and spinal cord, including the hypothalamic paraventricular nucleus (PVN). In this study, we examined the effects of intracerebroventricular (i.c.v.) administration of NPFF on activation of chemically identified PVN neurones that project to the brainstem nucleus of the solitary tract (NTS). In conscious rats, i.c.v. NPFF at a dose of 10 micro g, but not 8 micro g, caused an increase in arterial blood pressure. Immunohistochemical analysis revealed a dose-dependent increase in activated (Fos positive) PVN neurones following i.c.v. NPFF administration compared to controls receiving i.c.v. saline. Activated PVN neurones were located predominantly in the parvocellular compartment of the nucleus with relatively few Fos positive cells in the magnocellular subdivision. Chemical identification of activated neurones revealed significant number of activated cells to be oxytocin positive, whereas only few vasopressin, tyrosine hydroxylase (TH) or corticotrophin-releasing factor (CRF) neurones were double-labelled. Injection of the retrograde tracer fluorogold into the NTS resulted in labelling of significant numbers of parvocellular oxytocin, but not vasopressin, TH or CRF, PVN neurones. We conclude that centrally administered NPFF stimulates brainstem-projecting oxytocin PVN neurones. Oxytocin released from terminals within the NTS oxytocin thus modulate the activity of ascending visceral autonomic pathways that synapse initially within the NTS.     

The Anorectic Effect of Neuropeptide AF is Associated with Satiety-Related Hypothalamic Nuclei

Neuropeptide AF (NPAF), a member of the RFamide family, is encoded by the same gene as neuropeptide FF (NPFF), which causes short-term anorexia. However, reports on the role of NPAF on appetite-related process are lacking. Thus, i.c.v. injections of 4.0, 8.0 and 16.0 nmol NPAF were administered to chicks to observe its effect on food and water intake. Chicks treated with 8.0 and 16.0 nmol i.c.v. NPAF decreased both their food and water intake. Additionally, all doses of NPAF injected caused a similar reduction in whole blood glucose concentration 180 min after injection. In a second experiment, chicks that received i.c.v. NPAF had an increased number of c-Fos immunoreactive cells in the dorsomedial, paraventricular (magnocellular and parvicellular parts) and ventromedial nuclei. The arcuate nucleus and lateral hypothalamic area were not affected. In a third experiment, NPAF-treated chicks exhibited fewer feeding pecks and spent less time perching, whereas they spent an increased time in deep rest. Other behaviours, including exploratory pecking, escape attempts, defecations, distance moved, and time spent standing, sitting and preening, were not affected by NPAF injection. We conclude that NPAF causes anorectic effects that are associated with the hypothalamus.     

Beta-melanocyte-stimulating hormone potently reduces appetite via the hypothalamus in chicks

The melanocortin system together with other appetite-related systems plays a significant role in appetite regulation. The appetite-related effects of one such melanocortin, β-melanocyte-stimulating hormone (MSH), are well documented in rodents; however, its effects in the avian class are not thoroughly understood. Thus, we designed a study to determine the effects of i.c.v. β-MSH injection on food and water intake, plasma corticosterone concentration, ingestive and non-ingestive behaviours, and hypothalamic neuronal activation using Cobb-500 chicks. Chicks responded to β-MSH-treatment with a reduction in food and water intake; however when water intake was measured independently of food intake, it was not affected. β-MSH-treated chicks also had increased plasma corticosterone concentrations and increased c-Fos reactivity in the periventricular, paraventricular and infundibular nuclei, and the ventromedial hypothalamus; however, the lateral hypothalamus was not affected. The effect on food intake is primary because behaviours that may be competitive with food intake were not increased in β-MSH-treated chicks. Based on these results, we conclude that β-MSH causes anorexigenic effects that are likely primarily mediated via stimulation of satiety-related hypothalamic nuclei in broiler-type chicks.     

Short-term anorexigenic effects of central neuropeptide VF are associated with hypothalamic changes in chicks

The present study was designed to measure food and water intake, changes in hypothalamic chemistry, and other behaviour modifications after central injection of neuropeptide (NP) VF in broiler type chicks. In Experiment 1, chicks responded to central NPVF with a reduction in food intake for up to 90 min post injection. Water intake was unaffected. In Experiment 2, NPVF exerted a less potent and shorter duration of attenuated food intake than did the structurally related NPFF. In Experiment 3, 16.0 nmol NPVF reversed the prolactin-releasing peptide induced orexigenic effect. In Experiment 4, central NPVF treatment was associated with decreased c-Fos immunoreactivity in the lateral hypothalamus, whereas c-Fos immunoreactivity in the dorsomedial nucleus, infundibular nucleus (homologue to the mammalian arcuate nucleus) and ventromedial nucleus was increased. In Experiment 5, behaviours unrelated to ingestion including sit, stand, deep rest and locomotion were affected by central NPVF injection. Some of these behaviours are incompatible with ingestion and may contribute to hypothalamic associated perception of satiety after central NPVF. In conclusion, NVPF is a short-term regulator of appetite and its effects are associated with hypothalamic and behaviour changes in chicks.     

Distribution of the neuropeptide FF1 receptor (hFF1) in the human hypothalamus and surrounding basal forebrain structures: immunohistochemical study

Neuropeptides with C-terminal RFamide and their receptors NPFF1 (FF1) and NPFF2 (FF2) have been implicated in a wide variety of functions, including nociception and autonomic and neuroendocrine regulation. Recent studies indicate that the FF1, but not FF2, mRNA is highly expressed in the human hypothalamus. In the present study, localization of FF1 in the human hypothalamus and surrounding regions was studied immunohistochemically by using an antibody against human FF1 (hFF1). Brain sections from healthy 30-50-year-old individuals were used for hFF1 immunohistochemistry. The highest density of hFF1-stained cells was found in the posterior division of the bed nucleus of the stria terminalis and in the zona incerta. A moderate density of cells was observed in the perifornical nucleus, infundibular nucleus, tuberomammillary nucleus, and lateral tuberal nucleus. A lesser density was revealed in the dorsomedial hypothalamic nucleus, basal nucleus of Meynert, and anterior amygdaloid area. Only scattered hFF1 cells were found in the suprachiasmatic nucleus and hypothalamic paraventricular nucleus. hFF1 cells and fibers were absent in the supraoptic and mammillary nuclei. Single and double strands of hFF1-immunopositive punctate varicosities marked cellular processes of different caliber. The density of hFF1-immunostained fiber networks did not always coincide with that of hFF1-immunostained cells. hFF1 immunoreactivity was also found in the wall of blood vessels within most brain areas studied. Localization of hFF1 in discrete regions of the hypothalamus and extended amygdala may provide important insights into the role of amidated neuropeptides in central autonomic and neuroendocrine control in the human brain.