Neuronal interaction between melanin-concentrating hormone- and alpha-melanocyte-stimulating hormone-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.     

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.     

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.     

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.).     

Leptins and leptin receptor expression in the goldfish (Carassius auratus). Regulation by food intake and fasting/overfeeding conditions

Leptin is a hormone involved in feeding and body weight regulation in vertebrates, but the relationship between energy status and leptin has not been clearly established in fish. The aim of this study was to investigate in a teleost, the goldfish (Carassius auratus), the tissue expression pattern of two leptins (gLep-aI and gLep-aII) and leptin receptor (gLepR); and the effect of feeding on expression of these genes. Leptin system expression in goldfish was firstly analyzed in fish under overfeeding (2 weeks) or fasting (1 week), and secondly, at different postfeeding times (0, 3, 6, 9 and 12 h). Goldfish has two Lep-a paralog genes, gLep-aI was widely expressed in central and peripheral tissues, whereas gLep-aII was preferentially expressed in brain. This different distribution pattern of leptins suggests that they can play different physiological roles in goldfish. The gLepR mRNA was ubiquitous expressed, with the highest expression in the telencephalon and hypothalamus. No significant differences in the leptin system expression were found among control, overfed and fasting groups, suggesting an apparent lack of correlation between nutritional status and leptin system in goldfish. Hepatic expression of gLep-aI significantly increased 9 h after feeding time, while hypothalamic leptin system expression did not change after feeding. In summary, leptin in goldfish could signal short-term changes in food intake, as postprandial satiety, but seems to be independent of fasting/overfeeding conditions in this teleost. The widespread distribution of leptins and leptin receptor in goldfish strongly supports that this hormone may have pleitropic actions in fish.     

Role of the melanin-concentrating hormone neuropeptide in sleep regulation

Melanin-concentrating hormone (MCH), a neuropeptide secreted by a limited number of neurons within the tuberal hypothalamus, has been drawn in the field of sleep only fairly recently in 2003. Since then, growing experimental evidence indicates that MCH may play a crucial role in the homeostatic regulation of paradoxical sleep (PS). MCH-expressing neurons fire specifically during PS. When injected icv MCH induces a 200% increase in PS quantities in rats and the lack of MCH induces a decrease in sleep quantities in transgenic mice. Here, we review recent studies suggesting a role for MCH in the regulation of the sleep–wake cycle, in particular PS, including insights on (1) the specific activity of MCH neurons during PS; (2) how they might be controlled across the sleep–wake cycle; (3) how they might modulate PS; (4) and finally whether MCH might take part in the expression of some symptoms observed in primary sleep disorders.     

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.     

Central and peripheral effects of ghrelin on energy balance, food intake and lipid metabolism in teleost fish

Ghrelin was first identified and characterized from rat stomach as an endogenous ligand for the growth hormone secretagogue receptor. Ghrelin and its receptor system are present not only in peripheral tissues such as stomach and intestine, but also in the central nervous system of mammals. Interestingly, administration of ghrelin induces an orexigenic effect and also modifies locomotor activity, suggesting its involvement in feeding control and the regulation of energy balance, in addition to the regulation of growth hormone release. Information about ghrelin in non-mammals, such as teleost fish, has also been increasing, and important data have been obtained. An understanding of the evolutionary background of the energy regulation system and the central and peripheral roles of ghrelin in teleost fish could provide indications as to their roles in mammals, particularly humans. In this review, we overview the central and peripheral effects of ghrelin on energy balance, locomotor activity, and lipid metabolism in teleost fish.     

Time-dependent effects of leptin on food intake and locomotor activity in goldfish

The present study investigates the possible circadian dependence of leptin effects on food intake, locomotor activity, glycemia and plasma cortisol levels in goldfish (Carassius auratus). Fish were maintained under 12L:12D photoperiod and subjected to two different feeding schedules, one group fed during photophase (10:00) and the other one during scotophase (22:00). Leptin or saline were intraperitoneally injected at two different times (10:00 or 22:00), coincident or not with the meal time. To eliminate the entraining effect of the light/dark cycle, goldfish maintained under 24 h light (LL) were fed and leptin-injected at 10:00. A reduction in food intake and locomotor activity and an increase in glycemia were found in goldfish fed and leptin-injected at 10:00. No significant changes in circulating cortisol were observed. Those effects were not observed when leptin was administered during the scotophase, regardless the feeding schedule; neither in fish maintained under LL, suggesting that a day/night cycle would be necessary to observe the actions of leptin administered during the photophase. Changes in locomotor activity and glycemia were only observed in goldfish when leptin was injected at daytime, coincident with the feeding schedule, suggesting that these leptin actions could be dependent on the feeding time as zeitgeber. In view of these results it appears that the circadian dependence of leptin actions in goldfish can be determined by the combination of both zeitgebers, light/dark cycle and food. Our results point out the relevance of the administration time when investigating regulatory functions of hormones.     

Regulation of food intake by acyl and des-acyl ghrelins in the goldfish

Our recent research has indicated that intracerebroventricular (i.c.v.) and intraperitoneal (i.p.) administration of n-octanoic acid-modified ghrelin (acyl ghrelin) stimulates food intake and locomotor activity in the goldfish. The manner in which peripherally administered acyl ghrelin regulates food intake, however, remains unclear. In contrast to acyl ghrelin, non-acylated ghrelin (des-acyl ghrelin) does not exert an orexigenic action or induce hypermotility. To this extent, the biological role of des-acyl ghrelin in fish is unknown. Given the possible involvement of afferent pathways in mediating the effects of acyl ghrelin, as is known to occur in rodents, we examined the effect of capsaicin, a neurotoxin which destroys primary sensory (vagal and splanchnic) afferents, on the orexigenic activity induced by i.p.-injected acyl ghrelin. Pretreatment with i.p.-injected capsaicin (0.16 micromol/g body weight (BW)) cancelled the orexigenic action of i.p.-injected acyl ghrelin (8 pmol/g BW), although i.p.-injected capsaicin alone did not affect food intake. The effect of des-acyl ghrelin on the orexigenic action of acyl ghrelin in the goldfish was also investigated. The i.c.v. and i.p. injection of des-acyl ghrelin at doses 3-10 times higher than that of acyl ghrelin suppressed the orexigenic action of i.c.v.- and i.p.-injected acyl ghrelin (doses of 1 and 8 pmol/g BW). In contrast, injection of des-acyl ghrelin alone did not show any inhibitory effect on food intake. These results suggest that, as is seen in rodents, circulating acyl ghrelin derived from peripheral tissues acts via primary sensory afferent pathways on feeding centers in the brain. The results also show that des-acyl ghrelin inhibits acyl ghrelin-induced orexigenic activity in goldfish.     

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 in the goldfish by interaction between ghrelin and orexin

Intracerebroventricular (ICV) administration of ghrelin, orexin and neuropeptide Y (NPY) stimulates food intake in goldfish. Orexin and NPY interact with each other in the regulation of feeding, while ghrelin-induced feeding has also shown to be mediated by NPY in the goldfish model. To investigate the interaction between ghrelin and orexin, we examined the effects of a selective orexin receptor-1 antagonist, SB334867, and a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]-GHRP-6, on ghrelin- and orexin-A-induced feeding. Ghrelin-induced food intake was completely inhibited for 1h following ICV preinjection of SB334867, while [D-Lys(3)]-GHRP-6 attenuated orexin-A stimulated feeding. Furthermore, ICV administration of ghrelin or orexin-A at a dose sufficient to stimulate food intake increased the expression of each other's mRNA in the diencephalon. These results indicate that, in goldfish, ghrelin and orexin-A have interacting orexigenic effects in the central nervous system. This is the first report that orexin-A-induced feeding is mediated by the ghrelin signaling in any animal model.     

Development of food intake controls: Neuroendocrine and environmental regulation of food intake during early life

This article is part of a Special Issue "Energy Balance".     

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.     

Brain lesions and short-term endocrine effects of monosodium L-glutamate in goldfish,Carassius auratus

Summary Monosodium L-glutamate (MSG) was injected intraperitoneally into goldfish at a dosage of 2.5mg/g body weight. At 24 h post-injection there was a marked hypertrophy and edema in the region of the nucleus lateralis tuberis (NLT) from the anterior margin of the pituitary stalk through to the posterior end of the NLT, irrespective of the sex of the goldfish. A similar hypertrophy and edema occurred ventral to the anterior commissure in the preoptic region in the anterior-ventral nucleus preopticus periventricularis (NPP). At 6 h post-injection a slight vacuolization was evident in these two regions, and at two days the hypertrophy and edema had abated from the extent observed at 24 h post-injection. At five and eight days post-injection only necrotic cells were found in the affected NLT region, but only a small band of necrotic cells was evident in the anterior-ventral preoptic region. No other brain lesions were evident. Serum levels of gonadotropin (GtH) were increased at 6 h, 24 h, and two days after treatment with MSG, but were similar to control values at five, seven and eight days after MSG in male and female goldfish. Exocytosis of small dark secretory granules in gonadotrophs was evident at 24 h after MSG in a fish with a somewhat greater increase in serum GtH than usually found. The time course of increased serum GtH levels postinjection of MSG is consistent with the observed time course of hypertrophy and atrophy of NLT neurons; the increase in serum levels of GtH is interpreted to reflect a stimulation of release of GtH-releasing factor from neurons in the NLT. Electron microscope investigation indicates that prolactin cells have increased secretory and synthetic activity from 24 h through to seven days post-injection of MSG. The mechanism for stimulation of the prolactin cells by MSG is not known. No other changes in activity of adenohypophysial secretory cells were found.Supported by grant A-6371 from the Natural Sciences and Engineering Research Council of CanadaCNRS France and NRC Canada Exchange Scientist.     

Origin of the pituitary innervation in the goldfish

Despite the large number of studies devoted to the pituitary of teleosts, the origin of the direct pituitary innervation is still largely unknown. Although such a model is ideal for applying retrograde transport techniques, these methods involve the difficult in vivo injection of tracers into the pituitary and have never been applied. Recently, a lipophilic fluorescent dye (1-1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanin; DiI) has been introduced and shown to have the capacity of being transported by the membranes of paraformaldehydefixed tissues. Microcrystals of DiI were implanted via a ventral approach into the pituitary of goldfish previously fixed by intracardiac perfusion of paraformaldehyde. The goldfish heads were kept immersed in paraformaldehyde for various periods of time (2–6 weeks). The brains were then dissected and cut transversally using a Vibratome. The results demonstrate that hypophysiotrophic areas are essentially restricted to the preoptic region, the mediobasal hypothalamus and the nucleus dorsolateralis thalami. In addition, cell bodies probably containing gonadotrophin releasing-hormone were also retrogradely stained along a pathway that can be traced up to the olfactory bulbs. The results also confirm that cell bodies, located around the ventral aspect of the preoptic recess and probably corresponding to dopaminergic neurons, project to the pituitary. Large neurons have also been observed in the rostral dorsal midbrain tegmentum just caudal to the posterior commissure. Most neurons of the so-called paraventricular organ remain unstained. Finally, a fiber tract originating from an undetermined territory of the posterior brain has been observed. The results are discussed in relation to the possible chemical nature of the hypophysiotropic neurons.