Feeding-induced changes of melanin-concentrating hormone (MCH)-like immunoreactivity in goldfish brain

Intracerebroventricular (ICV) injection of melanin-concentrating hormone (MCH) influences feeding behavior in the goldfish and exerts an anorexigenic action in goldfish brain, unlike its orexigenic action in mammals. Despite a growing body of knowledge concerning MCH function in mammals, the role of MCH in appetite has not yet been well studied in fish. The aim of the present study was to investigate the involvement of endogenous MCH in the feeding behavior of the goldfish. We examined the distribution of MCH-like immunoreactivity (MCH-LI) in the goldfish brain and the effect of feeding status upon this distribution. Neuronal cell bodies containing MCH-LI were localized specifically to four areas of the hypothalamus. Nerve fibers with MCH-LI were found mainly in the neurohypophysis, with a few in the telencephalon, mesencephalon, and diencephalon. The number of neuronal cell bodies containing MCH-LI in the dorsal area adjoining the lateral recess of the third ventricle in the posterior and inferior lobes of the hypothalamus showed a significant decrease in fasted fish compared with that in normally fed fish, although other areas showed no evident differences. We also administered an antiserum against fish MCH (anti-MCH serum) by ICV injection and examined its immunoneutralizing effect on food intake by using an automatic monitoring system. Cumulative food intake was significantly increased by ICV injection of the anti-MCH serum. These results indicate that MCH potentially functions as an anorexigenic neuropeptide in the goldfish brain, and that the further study of the evolutionary background of the MCH system and its role in appetite is warranted. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (K.M. and A.T.) and by a research grant from the Toyama Marine Biotechnology Association (K.M.).     

Interactions between gonadotropin-releasing hormone (GnRH) and orexin in the regulation of feeding and reproduction in goldfish (Carassius auratus)

Links between energy homeostasis and reproduction have been demonstrated in vertebrates. As a general rule, abundant food resources favor reproduction whereas low food availability induces an inhibition of reproductive processes. In both mammals and fish, gonadotropin-releasing hormone (GnRH) and orexin (OX) are hypothalamic neuropeptides that play critical roles in the regulation of sexual behavior and appetite, respectively. In order to assess possible interactions between orexin and GnRH in the control of feeding and reproduction in goldfish, we examined the effects of chicken GnRH (cGnRH-II) intracerebroventricular (ICV) injection on feeding behavior and OX brain mRNA expression as well as the effects of orexin ICV injections on spawning behavior and cGnRH-II brain mRNA expression. Treatment with cGnRH-II at doses that stimulate spawning (0.5 ng/g or 1 ng/g) resulted in a decrease in both food intake and hypothalamic orexin mRNA expression. Treatment with orexin A at doses that stimulate feeding (10 ng/g) induced an inhibition of spawning behavior and a decrease in cGnRH-II expression in the hypothalamus and optic tectum-thalamus. Our results suggest that the anorexigenic actions of cGnRH-II in goldfish might be in part mediated by OX and that orexin inhibits reproductive behavior in part via the inhibition of the GnRH system. Our data suggest the existence of a coordinated control of feeding and reproduction by the orexin and GnRH systems in goldfish.     

Regulation of food intake by neuropeptide Y in goldfish

In mammals, neuropeptide Y (NPY) is a potent orexigenic factor. In the present study, third brain ventricle (intracerebroventricular) injection of goldfish NPY (gNPY) caused a dose-dependent increase in food intake in goldfish, and intracerebroventricular administration of NPY Y1-receptor antagonist BIBP-3226 decreased food intake; the actions of gNPY were blocked by simultaneous injection of BIBP-3226. Goldfish maintained on a daily scheduled feeding regimen display an increase in NPY mRNA levels in the telencephalon-preoptic area and hypothalamus shortly before feeding; however, a decrease occured in optic tectum-thalamus. In both fed and unfed fish, brain NPY mRNA levels decreased after scheduled feeding. Restriction in daily food ration intake for 1 wk or food deprivation for 72 h resulted in increased brain NPY mRNA levels. Results from these studies demonstrate that NPY is a physiological brain signal involved in feeding behavior in goldfish, mediating its effects, at least in part, through Y1-like receptors in the brain.     

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na(+)-K(+)-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na(+)-K(+)-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.     

Gonadotropin-releasing hormone II (GnRH II) mediates the anorexigenic actions of α-melanocyte-stimulating hormone (α-MSH) and corticotropin-releasing hormone (CRH) in goldfish

Intracerebroventricular (ICV) administration of gonadotropin-releasing hormone II (GnRH II), which plays a crucial role in the regulation of reproduction in vertebrates, markedly reduces food intake in goldfish. However, the neurochemical pathways involved in the anorexigenic action of GnRH II and its interaction with other neuropeptides have not yet been identified. Alpha-melanocyte-stimulating hormone (α-MSH), corticotropin-releasing hormone (CRH) and CRH-related peptides play a major role in feeding control as potent anorexigenic neuropeptides in goldfish. However, our previous study has indicated that the GnRH II-induced anorexigenic action is not blocked by treatment with melanocortin 4 receptor (MC4R) and CRH receptor antagonists. Therefore, in the present study, we further examined whether the anorexigenic effects of α-MSH and CRH in goldfish could be mediated through the GnRH receptor neuronal pathway. ICV injection of the MC4R agonist, melanotan II (80 pmol/g body weight; BW), significantly reduced food intake, and its anorexigenic effect was suppressed by ICV pre-administration of the GnRH type I receptor antagonist, antide (100 pmol/g BW). The CRH-induced (50 pmol/g BW) anorexigenic action was also blocked by treatment with antide. ICV injection of CRH (50 pmol/g BW) induced a significant increase of the GnRH II mRNA level in the hypothalamus, while ICV injection of melanotan II (80 pmol/g BW) had no effect on the level of GnRH II mRNA. These results indicate that, in goldfish, the anorexigenic actions of α-MSH and CRH are mediated through the GnRH type I receptor-signaling pathway, and that the GnRH II system regulates feeding behavior.     

Corticotropin-releasing hormone mediates alpha-melanocyte-stimulating hormone-induced anorexigenic action in goldfish

alpha-Melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH) both suppress food intake, and the alpha-MSH- or CRH-signaling pathway has possible potency to mediate anorexigenic actions induced by most other neuropeptides in goldfish. Therefore, using specific receptor antagonists, we examined whether the anorexigenic actions of alpha-MSH and CRH mutually interact. The inhibitory effect of ICV injection of the alpha-MSH agonist, melanotan II (MT II), on food intake was abolished by treatment with a CRH 1/2 receptor antagonist, alpha-helical CRH((9-41)), whereas the anorexigenic action of ICV-injected CRH was not affected by treatment with a melanocortin 4 receptor antagonist, HS024. This led us to investigate whether alpha-MSH-containing neurons in the goldfish brain have direct inputs to CRH-containing neurons, using confocal laser scanning microscopy. alpha-MSH- and CRH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. alpha-MSH-containing nerve fibers or endings lay in close apposition to CRH-containing neurons in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These results indicate that, in goldfish, alpha-MSH-induced anorexigenic action is mediated by the CRH-signaling pathway, and that CRH plays a crucial role in the regulation of feeding behavior as an integrated anorexigenic neuropeptide in this species.     

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na⁺-K⁺-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na⁺-K⁺-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.     

Relationship between anorexigenic action of pituitary adenylate cyclase-activating polypeptide (PACAP) and that of corticotropin-releasing hormone (CRH) in the goldfish, Carassius auratus

Our recent research has indicated that intracerebroventricular (ICV) injection of pituitary adenylate cyclase-activating polypeptide (PACAP) suppresses food intake and locomotor activity in the goldfish. However, the anorexigenic mechanism of PACAP has not yet been clarified. The aim of this study was to investigate the relationship between the anorexigenic action of PACAP and that of corticotropin-releasing hormone (CRH), which is implicated in the regulation of energy homeostasis as a powerful anorexigenic peptide in the goldfish brain. We first examined feeding-induced changes in the expression of CRH mRNA, and the effect of ICV administration of PACAP on the expression of CRH mRNA in the goldfish brain. Semiquantitative analysis revealed that the expression of CRH mRNA was significantly increased by excessive feeding for 7 days. ICV administration of PACAP at a dose sufficient to suppress food intake induced a significant increase in the expression of CRH mRNA. We also examined the effect of alpha-helical CRH(9-41), a CRH antagonist, on the anorexigenic action of PACAP in the goldfish. The inhibitory effect of PACAP was completely suppressed by treatment with alpha-helical CRH(9-41). We finally investigated the effect of ICV-administered CRH on locomotor activity in the goldfish. CRH at a dose sufficient to suppress food intake induced a significant increase in locomotor activity, unlike ICV-injected PACAP. These results suggest that, in the goldfish, the anorexigenic action of PACAP is related to the CRH neuronal pathway, but that the modulation of locomotor activity by PACAP is independent of modulation by CRH.     

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus

Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45-60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.     

Epithelial remodeling and claudin mRNA abundance in the gill and kidney of puffer fish (<Emphasis Type="Italic">Tetraodon biocellatus</Emphasis>) acclimated to altered environmental ion levels

In water of varying ion content, the gills and kidney of fishes contribute significantly to the maintenance of salt and water balance. However, little is known about the molecular architecture of the tight junction (TJ) complex and the regulation of paracellular permeability characteristics in these tissues. In the current studies, puffer fish (Tetraodon biocellatus) were acclimated to freshwater (FW), seawater (SW) or ion-poor freshwater (IPW) conditions. Following acclimation, alterations in systemic endpoints of hydromineral status were examined in conjunction with changes in gill and kidney epithelia morphology/morphometrics, as well as claudin TJ protein mRNA abundance. T. biocellatus were able to maintain endpoints of hydromineral status within relatively tight limits across the broad range of water ion content examined. Both gill and kidney tissue exhibited substantial alterations in morphology as well as claudin TJ protein mRNA abundance. These responses were particularly pronounced when comparing fish acclimated to SW versus those acclimated to IPW. TEM observations of IPW-acclimated fish gills revealed the presence of cells that exhibited the typical characteristics of gill mitochondria-rich cells (e.g. voluminous, Na⁺-K⁺-ATPase-immunoreactive, exposed to the external environment at the apical surface), but were not mitochondria-rich. To our knowledge, this type of cell has not previously been described in hyperosmoregulating fish gills. Furthermore, modifications in the morphometrics and claudin mRNA abundance of kidney tissue support the notion that spatial alterations in claudin TJ proteins along the nephron of fishes will likely play an important role in the regulation of salt and water balance in these organisms.     

Ion-poor water and dietary salt deprivation upregulate the ghrelinergic system in the goldfish (Carassius auratus)

Because the ghrelinergic system in teleost fishes is broadly expressed in organs that regulate appetite as well as those that contribute to the regulation of salt and water balance, we hypothesized that manipulating salt and water balance in goldfish (Carassius auratus) would modulate the ghrelinergic system. Goldfish were acclimated to either freshwater (FW) or ion-poor FW (IPW) and were fed either a control diet containing 1% NaCl or low-salt diet containing 0.1% NaCl. Endpoints of salt and water balance, i.e., serum Na⁺ and Cl⁻ levels, muscle moisture content and organ-specific Na⁺-K⁺-ATPase (NKA) activity, were examined in conjunction with brain, gill and gut mRNA abundance of preproghrelin and its receptor, growth hormone secretagogue receptor (ghs-r). Acclimation of fish to IPW reduced serum osmolality and Cl⁻ levels and elevated kidney NKA activity, while FW fish fed a low NaCl diet exhibited a modest reduction in muscle moisture content but otherwise no apparent osmoregulatory disturbance. In contrast, a combined treatment of IPW acclimation and low dietary NaCl content reduced serum osmolality and Cl⁻ levels, elevated muscle moisture content and increased gill, kidney and intestinal NKA activity. This intensified response to the combined effects of water and dietary ion deprivation is consistent with an increased effort to enhance ion acquisition. In association with these latter observations, a significant upregulation of preproghrelin mRNA expression in brain and gut was observed. A significant increase in ghs-r mRNAs was also observed in the gill of goldfish acclimated to IPW alone but a reduction in dietary NaCl content did not impact the ghrelinergic system of goldfish in FW. The results support the hypothesis that the ghrelinergic system is modulated in response to manipulated salt and water balance. Whether the central and peripheral ghrelinergic system contributes to ionic homeostasis in goldfish currently remains unclear and warrants further research.     

Regulation of food intake by melanin-concentrating hormone in goldfish

Melanin-concentrating hormone (MCH), originally discovered in the teleost pituitary, is a hypothalamic neuropeptide involved in the regulation of body color in fish. Although MCH is also present in the mammalian brain, it has no evident function in providing pigmentation. Instead, this peptide is now recognized to be one of the key neuropeptides that act as appetite enhancers in mammals such as rodents and primates. Although there has been little information about the central action of MCH on appetite in fish, recent studies have indicated that, in goldfish, MCH acts as an anorexigenic neuropeptide, modulating the α-melanocyte-stimulating hormone signaling pathway through neuronal interaction. These observations indicate that there may be major differences in the mode of action of MCH between fish and mammals. This paper reviews what is currently known about the regulation of food intake by MCH in fish, especially the goldfish.     

Molecular, cellular and physiological evidences for the anorexigenic actions of nesfatin-1 in goldfish

Background

Nesfatin-1 is a recently discovered anorexigen encoded in the precursor peptide, nucleobindin-2 (NUCB2) in mammals. To date, nesfatin-1 has not been described in any non-mammalian species, although some information is available in the sequenced genomes of several species. Our objective was to characterize nesfatin-1 in fish.

Methodology/Principal Findings

In the present study, we employed molecular, immunohistochemical, and physiological studies to characterize the structure, distribution, and appetite regulatory effects of nesfatin-1 in a non-mammalian vertebrate. A very high conservation in NUCB2 sequences, especially in the nesfatin-1 region was found in lower vertebrates. Abundant expression of NUCB2 mRNA was detected in several tissues including the brain and liver of goldfish. Nesfatin-1-like immunoreactive cells are present in the feeding regulatory nucleus of the hypothalamus and in the gastrointestinal tract of goldfish. Approximately 6-fold increase in NUCB2 mRNA levels was found in the liver after 7-day food-deprivation, and a similar increase was also found after short-term fasting. This points toward a possible liver specific role for NUCB2 in the control of metabolism during food-deprivation. Meanwhile, ∼2-fold increase at 1 and 3 h post-feeding and an ∼3-fold reduction after a 7-day food-deprivation was observed in NUCB2 mRNA in the goldfish hypothalamus. In vivo, a single intraperitoneal injection of the full-length native (goldfish; gf) nesfatin-1 at a dose of 50 ng/g body weight induced a 23% reduction of food intake one hour post-injection in goldfish. Furthermore, intracerebroventricular injection of gfnesfatin-1 at a dose of 5 ng/g body weight resulted in ∼50% reduction in food intake.

Conclusions/Significance

Our results provide molecular, anatomical and functional evidences to support potential anorectic and metabolic roles for endogenous nesfatin-1 in goldfish. Collectively, we provide novel information on NUCB2 in non-mammals and an anorexigenic role for nesfatin-1 in goldfish.     

Inhibitory effect of chicken gonadotropin-releasing hormone II on food intake in the goldfish, Carassius auratus

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, which possesses some structural variants. A molecular form known as chicken GnRH II ([His⁵ Trp⁷ Tyr⁸] GnRH, cGnRH II) is widely distributed in vertebrates, and has recently been implicated in the regulation of sexual behavior and food intake in an insectivore, the musk shrew. However, the influence of cGnRH II on feeding behavior has not yet been studied in model animals such as rodents and teleost fish. In this study, therefore, we investigated the role of cGnRH II in the regulation of feeding behavior in the goldfish, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV-injected cGnRH II at graded doses, from 0.1 to 10 pmol/g body weight (BW), induced a decrease of food consumption in a dose-dependent manner during 60 min after treatment. Cumulative food intake was significantly decreased by ICV injection of cGnRH II at doses of 1 and 10 pmol/g BW during the 60-min post-treatment observation period. ICV injection of salmon GnRH ([Trp⁷ Leu⁸] GnRH, sGnRH) at doses of 0.1-10 pmol/g BW did not affect food intake. The anorexigenic action of cGnRH II was completely blocked by treatment with the GnRH type I receptor antagonist, Antide. However, the anorexigenic action of cGnRH II was not inhibited by treatment with the corticotropin-releasing hormone (CRH) 1/2 receptor antagonist, α-helical CRH₍₉₋₄₁₎, and the melanocortin 4 receptor antagonist, HS024. These results suggest that, in the goldfish, cGnRH II, but not sGnRH, acts as an anorexigenic factor, as is the case in the musk shrew, and that the anorexigenic action of cGnRH II is independent of CRH- and melanocortin-signaling pathways.     

Interactions between orexin A, NPY and galanin in the control of food intake of the goldfish, Carassius auratus

The neuropeptides orexin A (OXA), neuropeptide Y (NPY) and galanin (GAL) have been shown to play a role in the regulation of food intake in mammals. They also significantly stimulate feeding in goldfish. In order to assess the interactions between these peptides in the control of feeding in goldfish, we investigated the effects of central injection of specific receptor antagonists for NPY (BIBP 3226) and GAL (M40) on OXA-induced feeding and the effects of desensitization of orexin receptors on NPY- and GAL-induced feeding. We investigated the effects of BIBP 3226 on GAL-induced feeding and the effects of M40 on NPY-induced feeding. We also examined the effects of coinjection of each pair of neuropeptides on feeding behavior. Injections of 10 ng/g OXA, 5 ng/g NPY and 10 ng/g GAL each induced an increase in feeding. Fish treated with 5 ng/g BIBP or 20 ng/g M40 had food consumption similar to saline controls. BIBP at 5 ng/g significantly reduced NPY- and OXA-induced feeding. Injections of 20 ng/g M40 significantly decreased GAL-induced feeding, but had no effect on OXA-induced feeding. Blocking of orexin receptors by treatment with high doses of OXA (100 ng/g) resulted in a decrease in both NPY- and GAL-induced feeding. Coinjection with 0.5 ng/g OXA and either 0.5 ng/g NPY or 0.5 ng/g GAL resulted in a food intake higher than that observed in saline control fish and in fish treated with NPY or GAL alone at 0.5 ng/g. NPY mRNA expression was increased in the telencephalon and in the hypothalamus compared to saline-treated fish, following injection of OXA. These results indicate that both NPY and GAL are at least, in part, dependent on coaction with OXA for the stimulation of food intake and feeding behavior in goldfish. In addition, the effects of OXA are mediated, in part, by the NPY pathway. This suggests a functional interdependence between these three peptidergic systems in the control of energy balance in goldfish.     

Trade-offs between feeding and shock avoidance in goldfish (Carassius auratus)

Previous work showed that goldfish learn to avoid a region of an aquarium where they receive a discrete shock to the flank. This avoidance is reduced if another fish is present next to the shock zone, suggesting fish trade-off avoidance against a tendency to associate [Dunlop, R., Millsopp, S., Laming, P., 2006. Avoidance learning in goldfish (Carassius auratus) and trout (Oncorhynchus mykiss) and implications for pain perception. App. Anim. Behav. Sci. 97 (2–4), 255–271]. Only shock intensity was adjusted in those experiments, here we report results where a requirement for food was also altered.Fish were trained to feed at a region of the aquarium where subsequently they would receive a discrete shock to the flank. One experimental group had different shock intensities applied, whereas in the other group shock intensity was consistent but food deprivation was varied.We show the number of feeding attempts and time spent in the feeding/shock zone decreased with increased shock intensity and with increased food deprivation the number and the duration of feeding attempts increased as did escape responses as this zone was entered. These data suggest that goldfish balance their need for food against avoidance of an acute noxious stimulus.     

Anorectic actions of prolactin-releasing peptide are mediated by corticotropin-releasing hormone receptors

Prolactin-releasing peptide (PrRP) reduces food intake and body weight and modifies body temperature when administered centrally in rats, suggesting a role in energy homeostasis. However, the mediators of PrRP's actions are unknown. The present study, therefore, first examined the possible involvement of the anorectic neuropeptides corticotropin-releasing hormone (CRH) and the melanocortins (e.g., alpha-melanocyte-stimulating hormone) in PrRP's effects on food intake and core body temperature and, second, determined if PrRP affects energy expenditure by measuring oxygen consumption (Vo2). Intracerebroventricular injection of PrRP (4 nmol) to 24-h-fasted male Sprague-Dawley rats decreased food intake and modified body temperature. Blockade of central CRH receptors by intracerebroventricular coadministration of the CRH receptor antagonist astressin (20 microg) reversed the PrRP-induced reduction in feeding. However, astressin's effect on PrRP-induced changes in body temperature was complicated because the antagonist itself caused a slight rise in body temperature. In contrast, intracerebroventricular coadministration of the melanocortin receptor-3/4 antagonist SHU-9119 (0.1 nmol) had no effect on any of PrRP's actions. Finally, intracerebroventricular injection of PrRP (4 nmol) caused a significantly greater Vo2 over a 3-h test period compared with vehicle-treated rats. These results show that the anorectic actions of PrRP are mediated by central CRH receptors but not by melanocortin receptors-3/4 and that PrRP can modify Vo2.     

Alpha-melanocyte-stimulating hormone mediates melanin-concentrating hormone-induced anorexigenic action in goldfish

In goldfish, intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits feeding behavior, and fasting decreases hypothalamic MCH-like immunoreactivity. However, while MCH acts as an anorexigenic factor in goldfish, in rodents MCH has an orexigenic effect. Therefore, we examined the involvement of two anorexigenic neuropeptides, alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH), in the anorexigenic action of MCH in goldfish, using an alpha-MSH receptor antagonist, HS024, and a CRH receptor antagonist, alpha-helical CRH((9-41)). ICV injection of HS024, but not alpha-helical CRH((9-41)), suppressed MCH-induced anorexigenic action for a 60-min observation period. We then examined, using a real-time PCR method, whether MCH affects the levels of mRNAs encoding various orexigenic neuropeptides, including neuropeptide Y (NPY), orexin, ghrelin and Agouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for NPY and ghrelin, but not for orexin and AgRP. These results indicate that the anorexigenic action of MCH in the goldfish brain is mediated by the alpha-MSH signaling pathway and is accompanied by inhibition of NPY and ghrelin synthesis.     

Thyrotropin Releasing Hormone (TRH) in goldfish (Carassius auratus): role in the regulation of feeding and locomotor behaviors and interactions with the orexin system and cocaine- and amphetamine regulated transcript (CART)

TRH is a peptide produced by the hypothalamus which major function in mammals is the regulation of TSH secretion by the pituitary. In fish, TRH does not appear to affect TSH secretion, suggesting that it might regulate other functions. In this study, we assessed the effects of central (intracerebroventricular, icv) injections of TRH on feeding and locomotor behavior in goldfish. TRH at 10 and 100 ng/g, but not 1 ng/g, significantly increased feeding and locomotor behaviors, as indicated by an increase in food intake and in the number of total feeding acts as compared to saline-injected fish. In order to assess possible interactions between TRH and other appetite regulators, we examined the effects of icv injections of TRH on the hypothalamic expression of orexin, orexin receptor and CART. The mRNA expression levels of all three peptides were significantly increased in fish injected with TRH at 100 ng/g as compared to saline-injected fish. Fasting increased TRH, orexin, and orexin receptor hypothalamic mRNA levels and decreased CART hypothalamic mRNA levels. Our results suggest that TRH is involved in the regulation of feeding/locomotor activity in goldfish and that this action is associated with a stimulation of both the orexin and CART systems.