LHRH-A and sGnRH-A were tested, in the presence or absence of pimozide (Pim), for their ability to stimulate increases in serum gonadotropin (GtH) levels and ovulation in prespawning female goldfish. In the absence of Pim, sGnRH-A was more active than LHRH-A in terms of stimulating serum GtH levels; neither peptide given alone was effective in stimulating ovulation. Pim potentiated the activity of both peptides; high dosages of either peptide, plus a high dosage of Pim, were highly effective in stimulating serum GtH and ovulation. A low dosage of sGnRH-A, plus a low dosage of Pim, were also effective in stimulating serum GtH and ovulation; however, a low dosage of LHRH-A plus a low dosage of Pim were ineffective. The time to ovulation following injections, for those treatments that were highly effective in inducing ovulation, was highly predictable, and oocyte fertility and viability were high. These results indicate that sGnRH-A, in the presence of Pim, is effective in stimulating ovulation in goldfish a...

In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain.

In mammals, ghrelin is a non-amidated peptide hormone, existing in both acylated and non-acylated forms, produced mainly from the X/A or ghrelin cells present in the mucosal layer of the stomach. Ghrelin is a natural ligand of the growth hormone (GH) secretagogue-receptor (GHS-R), and functions primarily as a GH-releasing hormone and an orexigen, as well as having several other biological actions. Among non-mammalian vertebrates, amino acid sequence of ghrelin has been reported in two species of cartilaginous fish, seven species of teleosts, two species of amphibians, one species of reptile and six species of birds. The structure and functions of ghrelin are highly conserved among vertebrates. This review presents a concise overview of ghrelin biology in non-mammalian vertebrates.

Different life history stages of juvenile Atlantic salmon around the period of smoltification were subjected to short term stress (confinement) over two sampling years using separate cohorts of hatchery-reared fish. In the first year, confinement stress suppressed subsequent feeding in smolts and post-smolts, but not parr, in association with elevated plasma cortisol levels. Post-smolts showed much lower levels of food intake than either parr or smolts. Plasma ghrelin levels were unaffected by stress in any group. In the second study year, pre-smolts had very low food intake and this was further suppressed following stress. Higher food intake levels in smolts were strongly inhibited post-stress. Both pre-smolts, and smolts showed elevated levels of plasma cortisol and glucose, and suppressed plasma ghrelin levels. Across both study years, stress that was accompanied by elevated plasma cortisol levels resulted in suppression of feeding, whereas a predicted association of suppressed plasma ghrelin levels with reduced food intake, was present only in the second year. The results of the present study indicate that elevated plasma cortisol is a consistent endocrine correlate of stress-suppression of feeding. It is also apparent that the peri-smolt period is associated with markedly reduced feeding in this stock of Atlantic salmon.

The biological actions of growth hormone (GH) are pleiotropic, including growth promotion, energy mobilization, gonadal development, appetite, and social behavior. Accordingly, the regulatory network for GH is complex and includes many endocrine and environmental factors. In fish, the neuroendocrine control of GH is multifactorial with multiple inhibitors and stimulators of pituitary GH secretion. In fish, GH release is under a tonic negative control exerted mainly by somatostatin. Sex steroid hormones and nutritional status influence the level of brain expression and effectiveness of some of these GH neuroendocrine regulatory factors, suggesting that their relative importance differs under different physiological conditions. At the pituitary level, some, if not all, somatotropes can respond to multiple regulators. Therefore, ligand- and function-specificity, as well as the integrative responses to multiple signals must be achieved at the level of signal transduction mechanisms. Results from investigations on a limited number of stimulatory and inhibitory GH-release regulators indicate that activation of different but convergent intracellular pathways and the utilization of specific intracellular Ca2+ stores are some of the strategies utilized. However, more work remains to be done in order to better understand the integrative mechanisms of signal transduction at the somatotrope level and the relevance of various GH regulators in different physiological circumstances.

In teleost fish, melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide released from the pituitary during white background adaptation. In the periphery MCH concentrates melanin granules in melanophores thus lightening the body color of fish. Evidence from mammalian studies has demonstrated the involvement of MCH in the control of energy balance and the reproductive axis. Information about the hormonal regulation of MCH neurons in non-mammalian systems is scarce and nothing is known about its role in the regulation of the reproductive axis. We here report the molecular characterization of two MCH precursors in the goldfish. Both precursors are peripherally expressed and the expression in the central nervous system is restricted to the mediobasal hypothalamus. Hypothalamic MCH-mRNA production is upregulated during white background adaptation. Both testosterone and estradiol stimulate MCH mRNA expression in the hypothalamus in a sex-dependent manner, with females showing the greatest responsiveness. In addition, in vitroexperiments demonstrated that graded doses of salmon MCH stimulate LH, but not GH, secretion from dispersed pituitary cells. Results suggest that hypothalamic MCH may participate in the steroid positive feedback loop on pituitary LH secretion.

A potential role for prolactin-releasing peptide (PrRP) in appetite regulation and hydromineral balance in goldfish was examined. PrRP was found to be expressed in discrete regions of the goldfish brain, in particular, the hypothalamus. Intraperitoneal (IP) or intracerebroventricular administration of PrRP had dose-dependent effects to suppress food intake in goldfish. Hypothalamic PrRP mRNA expression significantly increased after feeding, as well as after 7 days of food deprivation. Refeeding fish after 7 days food deprivation did not result in a postprandial increase in PrRP mRNA expression. These data suggest an anorexigenic role for PrRP in the short term around a scheduled meal time, but not over the longer term. IP injection of PrRP significantly increased pituitary prolactin (PRL) mRNA levels, suggesting involvement in the regulation of lactotroph activity. Acclimating goldfish to an ion-poor environment decreased serum osmolality and increased PrRP and PRL mRNA levels, providing evidence for PrRP involvement in hydromineral balance through its actions on lactotrophs. Acclimation to ion-poor water diminished the anorexigenic properties of PrRP in goldfish, indicating that a role for PrRP in goldfish satiation is counterbalanced by alternate systemic needs (i.e., osmoregulatory). This was further supported by an ability to reinstate the anorexigenic actions of PrRP in fish acclimated to ion-poor water by feeding a salt-rich diet. These studies provide evidence that PrRP is involved in regulating appetite and hydromineral balance in fish, and that the degree of involvement in either process varies according to overall systemic needs in response to environmental conditions.

Feeding behavior is a complex behavior that is closely associated with food intake. Fish have a wide variety of feeding habits and feeding patterns making them good experimental models for the study of the regulation of feeding behavior. The aquatic nature of fish often creates challenges in the study of feeding behavior and different approaches have been used by researchers, including field studies, observations of free-living animals, and laboratory experiments. Feeding behavior is regulated by a number of environmental factors and also by complex homeostatic mechanisms that involve central and peripheral hormonal factors as well as metabolites. This review summarizes our current knowledge on the control of feeding behavior of fish, with emphasis on the methodology used and on the endocrine and metabolic regulation of feeding.

In goldfish, growth hormone (GH) transiently rises 30 min after meals, returning to baseline at 1 h postmeal. Somatostatin (SRIF) is the major inhibitor of GH release. Three cDNAs encoding pre-pro-SRIF (PSS) have been previously cloned from goldfish brain: PSS-I, which encodes SRIF-14; PSS-II, which is potentially processed into gSRIF-28 that has [Glu1,Tyr7,Gly10]SRIF-14 at the COOH terminus; and PSS-III, which encodes [Pro2]SRIF-14 at its COOH terminus. In goldfish, bombesin (BBS), mimicking the endogenous gastrin-releasing peptide (GRP), acutely suppresses food intake and also stimulates GH release. Ghrelin was recently characterized in goldfish as a GH secretagogue and an orexigen. In this paper, we studied the changes in SRIF mRNA levels during feeding and analyzed the influences of BBS and ghrelin peptides on forebrain PSS expression. The results showed a 60% reduction in PSS-II mRNA after meals, but no changes in the expression of PSS-I and PSS-III were found. Intraperitoneal injections of 100 ng/g body wt of BBS increased GH secretion and decreased PSS-I and PSS-II gene expression. Intraperitoneal injection of goldfish ghrelin (100 ng/g body wt) transiently increased the serum GH levels and increased PSS-I, while decreasing PSS-II mRNA levels. Ghrelin (50 ng/g body wt) blocked the effects of BBS (100 ng/g body wt) on PSS-I but not on PSS-II expression. Coadministration of BBS and ghrelin decreased only the PSS-II gene expression. We conclude that the interactions between BBS/GRP and ghrelin can account for the postprandial variations in serum GH levels and the forebrain expression of PSS-II. Furthermore, we demonstrate that intraperitoneal administration of BBS reduces the ghrelin expression levels in the gut. Thus the inhibition of production of ghrelin in the gut may contribute to the satiety effects of BBS/GRP peptides.

This study analyzes daily changes in the expression of somatostatin precursors PSS-I and PSS-III (structurally related to cortistatin) in the goldfish brain. The results indicate that PSS-I expression correlates with the light cycle only in optic tectum-thalamus (OT-Tha). PSS-III expression correlates with the light cycle in telencephalon-preoptic area (Tel-POA) and OT-Tha. In Tel-POA, PSS-III reaches a minimum level at the beginning of the active phase and a maximum level late in this phase. These results suggest that PSS-I in OT-Tha and PSS-III in Tel-POA and OT-Tha could be involved in the control of the activity cycles in goldfish.

The brain, particularly the hypothalamus, integrates input from factors that stimulate (orexigenic) and inhibit (anorexigenic) food intake. In fish, the identification of appetite regulators has been achieved by the use of both peptide injections followed by measurements of food intake, and by molecular cloning combined with gene expression studies. Neuropeptide Y (NPY) is the most potent orexigenic factor in fish. Other orexigenic peptides, orexin A and B and galanin, have been found to interact with NPY in the control of food intake in an interdependent and coordinated manner. On the other hand cholecystokinin (CCK), cocaine and amphetamine-regulated transcript (CART), and corticotropin-releasing factor (CRF) are potent anorexigenic factors in fish, the latter being involved in stress-related anorexia. CCK and CART have synergistic effects on food intake and modulate the actions of NPY and orexins. Although leptin has not yet been identified in fish, administration of mammalian leptin inhibits food intake in goldfish. Moreover, leptin induces CCK gene expression in the hypothalamus and its actions are mediated at least in part by CCK. Other orexigenic factors have been identified in teleost fish, including the agouti-related protein (AgRP) and ghrelin. Additional anorexigenic factors include bombesin (or gastrin-releasing peptide), α-melanocyte-stimulating hormone (α-MSH), tachykinins, and urotensin I. In goldfish, nutritional status can modify the expression of mRNAs encoding a number of these peptides, which provides further evidence for their roles as appetite regulators: (1) brain mRNA expression of CCK, CART, tachykinins, galanin, ghrelin, and NPY undergo peri-prandial variations; and (2) fasting increases the brain mRNA expression of NPY, AgRP, and ghrelin as well as serum ghrelin levels, and decreases the brain mRNA expression of tachykinins, CART, and CCK. This review will provide an overview of recent findings in this field.

One of the most successful chromatic adaptations in vertebrates is the dorsal-ventral pigment pattern in which the dorsal skin is darkly colored, whereas the ventrum is light. In fish, the latter pattern is achieved because a melanization inhibition factor inhibits melanoblast differentiation and supports iridophore proliferation in the ventrum. In rodents, the patterned pigmentation results from regional production of the agouti-signaling protein (ASP). This peptide controls the switch between production of eumelanin and pheomelanin by antagonizing alphaMSH effects on melanocortin receptor (MCR) 1 in the melanocytes. In addition, ASP inhibits the differentiation and proliferation of melanoblast. Thus, the mammalian ASP may be homologous to the poikilotherm melanization inhibition factor. By screening of a genomic library, we deduced the amino acid sequence of goldfish ASP. The ASP gene is a four-exon gene spanning 3097 bp that encodes a 125-amino acid precursor. Northern blot analysis identified two different ASP mRNAs in ventral skin of red- and black-pigmented and albino fish, but no expression levels were observed in the dorsal skin of the same fish. The dorsal-ventral expression polarity was also detected in both black dorsally pigmented fish and albino fish. Pharmacological studies demonstrate that goldfish ASP acts as a melanocortin antagonist at Fugu MC1R and goldfish MC4R. In addition, goldfish ASP inhibited Nle4, D-Phe7-MSH-stimulated pigment dispersion in medaka melanophores. Our studies support agouti signaling protein as the melanization inhibition factor, a key factor in the development of the dorsal-ventral pigment pattern in fish.

In this study, we examined (i) the preprandial, postprandial and starvation-induced changes in the preproghrelin mRNA expression and serum ghrelin levels, and (ii) the effects of intracerebroventricular and intraperitoneal administration of ghrelin on food intake in goldfish. Slot blot analysis revealed a significant postprandial decrease in preproghrelin mRNA expression in the hypothalamus (1 and 3 h after feeding) and gut (3 h after feeding). A similar postprandial decrease (1 and 3 h after feeding) in serum ghrelin levels was also detected. In the fish that were unfed at the regular feeding time, the hypothalamic preproghrelin mRNA expression and the serum ghrelin levels remained unchanged, while the preproghrelin mRNA expression in the gut decreased 3 h after the regular feeding time. Starvation increased preproghrelin mRNA expression in the hypothalamus and gut on the 7th day. Serum ghrelin levels were significantly elevated on days 3 and 5 of starvation. Intracerebroventricular injections of n-octanoylated ghrelin-like peptides (gGRL[1–12]) (10 ng/g body weight) and human ghrelin (1 and 10 ng/g body weight) and intraperitoneal injections of n-octanoylated gGRL[1–12] (10 ng/g body weight), gGRL[1–19] (100 ng/g body weight) and human ghrelin (10 and 100 ng/g body weight) stimulated food intake in goldfish. The patterns of synthesis, secretion and actions indicate that ghrelin is an orexigen in goldfish.

In the present study the brain distribution of three somatostatin (SRIF)‐encoding genes, PSS‐I, PSS‐II, and PSS‐III, was analyzed by in situ hybridization (ISH) in the goldfish. The PSS‐I mRNA showed the widest distribution throughout the brain, whereas PSS‐II transcripts were restricted to some hypothalamic nuclei. On the other hand, PSS‐III presents an intermediate distribution pattern. All SRIF encoding genes are expressed in hypophysiotropic nuclei supporting the idea that, in addition to SRIF‐14, [Pro 2 ] SRIF‐14, and gSRIF‐28 have pituitary‐controlling functions. Moreover, each of the genes is expressed in nuclei directly associated with feeding behavior, suggesting a role for SRIF peptides in the central control of food intake and energy balance. Alternatively, they might have a role in processing sensory information related with feeding behavior, since PSS genes are expressed in the main gustatory, olfactory, and visual centers, which project to the hypothalamic feeding center in teleost fish. J. Comp. Neurol. 474:43–57, 2004. © 2004 Wiley‐Liss, Inc.

Although elevated plasma cortisol levels and a reduction in food intake are common features of the response to stress in fish, the potential role of cortisol in the regulation of food intake in these animals is poorly understood. In this study, goldfish (Carassius auratus) were fed ad libitum for 21 days diets prepared to contain 0 (Control), 50 (Low) or 500 (High) microg cortisol/g of food. While feeding remained unchanged in controls and in fish fed the High cortisol diet, daily food intake gradually increased in the Low cortisol diet group and was significantly elevated between days 9 and 21. At the end of the feeding trial, specific growth rate was lowest in fish fed the High cortisol diet, intermediate in those fed the Low cortisol diet, and highest in the controls. Feed conversion efficiency, on the other hand, was significantly reduced in both groups of fish fed the cortisol diets. After 3 weeks on the diets and relative to controls, the Low cortisol diet group was characterized by a 34% increase in neuropeptide Y (NPY) and a 22% decrease in corticotropin-releasing factor (CRF) mRNA levels in the telencephalon-preoptic brain region. In contrast, the High cortisol diet group was characterized by a 46% decrease in CRF mRNA levels and no significant change in NPY gene expression. In a separate experiment, intraperitoneal implants of cortisol (150 and 300 microg cortisol/g body weight) elicited a dose-dependent increase in NPY and decrease in CRF mRNA levels in the telencephalon-preoptic region at 72 h post-treatment. These results show that while moderate increases in plasma cortisol can stimulate food intake slowly over days, larger catabolic doses of glucocorticoids may mask the appetite-stimulatory effects of cortisol. Therefore, excess cortisol in goldfish can be associated with poor growth despite normal food intake. Furthermore, our results indicate that forebrain NPY and CRF may play a role in mediating the effects of cortisol on food intake in goldfish.

In many teleosts, the control of gonadotropin II (or luteinizing hormone) secretion is under the dual control of stimulatory and inhibitory neuroendocrine factors. The principal stimulating factor is gonadotropin-releasing hormone and the main inhibitor is dopamine. Inhibiting the activities of dopamine by antidopaminergic drugs potentiates the actions of exogenous gonadotropin-releasing hormone analogs, resulting in a surge release of luteinizing hormone and ovulation and spawning in a number of different species. As the effects of blocking the inhibitory actions of dopamine on gonadotroph cytology have not been studied, goldfish were treated with 2, 4, 6 or 8 injections of reserpine (0.1 mg/kg body weight), at 48 h intervals, and the numbers of gonadotrophic cells studied at 48 h following last injection. After two injections, the number of gonadotrophic cells increased by 189% over controls; after four injections the increase was 234%; after six injections the increase was 259% and after eight injections, 288%. The results suggest that dopamine has an inhibitory influence on the numbers of gonadotrophs.

In this paper, we report (i) the in situ localization, and (ii) meal time related and starvation induced changes in preprogalanin mRNA expression in the goldfish brain. The specific brain nuclei that express galanin mRNA are the area ventralis telencephali pars ventralis, nucleus preopticus periventricularis, nucleus lateralis tuberis, and the nucleus recessus lateralis. No changes in preprandial preprogalanin mRNA expression were found in the brain regions studied. No changes in postprandial preprogalanin mRNA expression were found compared to the preprandial levels. However, in unfed fish, a significant increase in preprogalanin mRNA expression was found in the telencephalon (1 and 3 h) and hypothalamus (3 h) after the scheduled feeding time. The postprandial preprogalanin mRNA expression in the telencephalon and hypothalamus of fed fish at 1 and 3 h were significantly lower than the mRNA expression levels in the unfed fish at the same time. Preprogalanin mRNA expression levels remain unaltered during 7 days of starvation. The presence of preprogalanin mRNA in brain nuclei involved in the regulation of food intake and pituitary hormone secretion suggests important physiological roles for galanin in goldfish.