Morio Azuma

Stimulatory effect of intracerebroventricular administration of orexin A on food intake in the zebrafish, Danio rerio

Orexin is a potent orexigenic neuropeptide implicated in feeding regulation of mammals. However, except for the case of goldfish, the involvement of orexin in the feeding behavior of teleost fish has not well been studied. Therefore, we investigated the role of orexin on food intake using a zebrafish (Danio rerio) model. We examined the effect of feeding status on orexin-like immunoreactivity and the expression level of orexin transcript in the brain. The number of neuronal cells showing orexin-like immunoreactivity in the hypothalamic region, including the posterior tuberal nucleus, was significantly increased in fish fasted for 7days. Orexin precursor mRNA levels in the brain obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of orexin A on food intake. Cumulative food intake was significantly increased by ICV administration of orexin A (at 0.3 and 3 pmol/g body weight, BW) during a 60-min observation period after treatment. The orexin A-induced orexigenic action (at 0.3 pmol/g BW) was blocked by treatment with an orexin receptor antagonist, SB334867, at 10 pmol/g BW. These results indicate that orexin A acts as feeding regulator in the zebrafish.

Neuropeptide Y stimulates food intake in the Zebrafish, Danio rerio.

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in mammals. However, except for the case of the goldfish, the involvement of NPY in the feeding behaviour of teleost fish has not well been studied. Therefore, we investigated the role of NPY in food intake using a zebrafish (Danio rerio) model because the molecular bases of NPY and its receptor have been well studied in this species. We examined the effect of feeding status on NPY‐like immunoreactivity and the expression level of the NPY transcript in the brain. The number of neuronal cells showing NPY‐like immunoreactivity in the hypothalamic regions, including the periventricular nucleus of posterior tuberculum and the posterior tuberal nucleus, was significantly increased in fish fasted for 7 days. NPY mRNA levels in the hypothalamus, but not the telencephalon, obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of i.c.v. administration of NPY on food intake. Cumulative food intake was significantly increased by i.c.v. administration of NPY (at 1 and 10 pmol/g body weight; BW) during a 60‐min observation period. The NPY‐induced orexigenic action (at 10 pmol/g BW) was blocked by treatment with a NPY Y1 receptor antagonist, BIBP‐3226, at 100 pmol/g BW. These results indicate that NPY acts as an orexigenic factor in the zebrafish.

Neuroendocrine control of feeding behavior and psychomotor activity by neuropeptideY in fish

Neuropeptide Y (NPY) is a neuropeptide distributed widely among vertebrates. In mammals, NPY and its related peptides such as pancreatic polypeptide and peptide YY (PYY) are distributed throughout the brain and gastrointestinal tissues, and are centrally involved in many physiological functions such as the regulation of food intake, locomotion and psychomotor activities through their receptors. With regard to non-mammalian vertebrates, there has also been intensive study aimed at the identification and functional characterization of NPY, PYY and their receptors, and recent investigations of the role of NPY have revealed that it exerts several behavioral effects in goldfish and zebrafish. Both of these species are excellent teleost fish models, in which it has been demonstrated that NPY increases food consumption as an orexigenic factor and reduces locomotor activity, as is the case in mammals. This paper reviews current knowledge of NPY derived from studies of teleost fish, as representative non-mammals, focusing particularly on the role of the NPY system, and examines its significance from a comparative viewpoint.

Orexin system in teleost fish.

Orexin is a neuropeptide distributed widely among vertebrates. In mammals, orexin and its receptor system are involved in the regulation of food intake, locomotion, and psychomotor activities including the sleep/wakefulness cycle. With regard to nonmammalian vertebrates, there has also been intensive study aimed at the identification and functional characterization of orexin and its receptor, and recent investigations of the role of orexin have revealed that it exerts behavioral effects in teleost fish. Goldfish and zebrafish are excellent teleost fish models, and in these species it has been demonstrated that orexin increases food consumption as an orexigenic factor and enhances locomotor activity, as well as being involved in the regulation of active and rest status (circadian rhythmicity and the sleep/wakefulness cycle), as is the case in mammals. This chapter reviews current knowledge of orexin derived from studies of teleost fish, as representative nonmammals, focusing particularly on the role of the orexin system, and examines its significance from a comparative viewpoint.

Gonadotropin-releasing hormone 2 suppresses food intake in the zebrafish, Danio rerio

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, of which several structural variants exist. A molecular form known as GnRH2 ([His5 Trp7 Tyr8]GnRH, also known as chicken GnRH II) is widely distributed in vertebrates except for rodents, and has recently been implicated in the regulation of feeding behavior in goldfish. However, the influence of GnRH2 on feeding behavior in other fish has not yet been studied. In the present study, therefore, we investigated the role of GnRH2 in the regulation of feeding behavior in a zebrafish model, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV injection of GnRH2 at 0.1 and 1 pmol/g body weight (BW) induced a marked decrease of food consumption in a dose-dependent manner during 30 min after feeding. Cumulative food intake was significantly decreased by ICV injection of GnRH2 at 1 pmol/g BW during the 30-min post-treatment observation period. The anorexigenic action of GnRH2 was completely blocked by treatment with the GnRH type I receptor antagonist Antide at 25 pmol/g BW. We also examined the effect of feeding condition on the expression level of the GnRH2 transcript in the hypothalamus. Levels of GnRH2 mRNA obtained from fish that had been provided excess food for 7 days were higher than those in fish that had been fed normally. These results suggest that, in zebrafish, GnRH2 acts as an anorexigenic factor, as is the case in goldfish.

Neuroendocrine control of feeding behavior and psychomotor activity by pituitary adenylate cyclase-activating polypeptide (PACAP) in vertebrates

Food intake is a fundamental for animals to surviving and keeping offspring. The hypothalamic region of the brain and the brain stem in vertebrates are a center that plays an important role in the control of feeding and its related behaviors including locomotor and psychomotor activities. Pituitary adenylate cyclase-activating polypeptide (PACAP) has firstly been identified as a hypophysiotropic hormone involved in adenohypophyseal hormone release, and subsequently has been considered as a neuropeptide exerting multifunctional roles in the central and peripheral nervous systems and several tissues in vertebrates. For example, PACAP is involved in the neuroendocrine control of food intake and acts as an anorexigenic peptide to regulate satiety. Recent works on animal models such as rodents and goldfish which are both excellent animal models for investigating the neuroendocrinological roles of PACAP have been extensively examined and considerable information has been accumulated. In addition, psychophysiological effects of PACAP on emotional behavior have recently been found. Therefore, this review article provides an overview of the neuroendocrine regulation of feeding behavior and psyphysiological activity by PACAP in vertebrates.

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/gBW). The CRH-induced (50 pmol/gBW) anorexigenic action was also blocked by treatment with antide. ICV injection of CRH (50 pmol/gBW) induced a significant increase of the GnRH II mRNA level in the hypothalamus, while ICV injection of melanotan II (80 pmol/gBW) 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.

The anorexigenic effect of cholecystokinin octapeptide in a goldfish model is mediated by the vagal afferent and subsequently through the melanocortin- and corticotropin-releasing hormone-signaling pathways.

We have been extensively investigating the mechanisms by which neuropeptides regulate feeding behavior by using a goldfish (Carassius auratus) model. In this species, the anorexigenic action of melanocortin peptide is centrally mediated via the corticotropin-releasing hormone (CRH)/CRH receptor neuronal system, whereas sulfated cholecystokinin octapeptide (CCK-8s) is involved in the appetite regulation as a peripheral anorexigenic factor. The aim of the present study was to identify the mechanism of the anorexigenic effect of peripherally injected CCK-8s, which has not yet been identified in goldfish. Co-administration of capsaicin, a neurotoxin that destroys primary sensory afferents, at 100 nmol/g BW, blocked the anorexigenic action of intraperitoneally injected CCK-8s (100 pmol/g BW), whereas the anorexigenic action of intracerebroventricularly injected CCK-8s (5 pmol/g BW) was not blocked by co-administration of capsaicin. Pre-treatment with a specific CRH receptor antagonist, α-helical CRH((9-41)), attenuated the anorexigenic action of CCK-8s. The expression level of CRH mRNA in the diencephalic tissue of the CCK-8s-injected group was not changed, but the level of proopiomelanocortin mRNA was significantly increased at 1h after treatment. Therefore, we have identified for the first time that the reduction of appetite induced by peripherally injected CCK-8s in goldfish appears to be mediated by the vagal afferent and subsequently through the melanocortin- and corticotropin-releasing hormone-signaling pathways.

The octadecaneuropeptide exerts an anxiogenic-like action in goldfish.

I.c.v. administration of the octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI), induces anorexigenic and anxiogenic-like actions in rodents. We have recently shown that, in goldfish, i.c.v. injection of ODN also reduces food consumption via the metabotropic endozepine receptor. However, there is little information regarding the structure of DBI and the psychophysiological roles of endozepines in fish. Therefore, in the present study, we isolated and cloned a cDNA encoding goldfish DBI. The deduced sequence exhibits high similarity with non-mammalian DBIs, and we investigated the effect of homologous ODN on psychomotor activity in goldfish. i.c.v. injection of synthetic goldfish ODN at 10 pmol/g body weight (BW) stimulated locomotor activity. Since intact goldfish placed in a tank with both black and white background areas prefers the black compartment, we developed a method for measuring the time taken for fish to move from the black to the white area. I.c.v. administration of diazepam (35 and 350 pmol/g BW) decreased, whereas i.c.v. administration of ODN (10 pmol/g BW) or the central-type benzodiazepine receptor inverse agonist FG-7142 (9 pmol/g BW) increased the time taken to move from the black to the white background area. The anxiogenic-like effect of ODN was blocked by the central-type benzodiazepine receptor antagonist flumazenil (100 pmol/g BW), but was not affected by the metabotropic endozepine receptor antagonist cyclo1-8[d-Leu(5)]octapeptide (100 pmol/g BW). These data indicate that ODN can potently affect locomotor and psychomotor activities in goldfish and that this action is mediated via the central-type benzodiazepine receptor-signaling pathway.

Pituitary adenylate cyclase-activating polypeptide induces somatolactin release from cultured goldfish pituitary cells

In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAP-induced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 10(-8) and 10(-7)M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC(1)R) antagonist, PACAP(6-38), at 10(-6)M, but not with the PACAP/VIP receptor antagonist, VIP(6-28). PACAP-induced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 10(-6)M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP(3))/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 10(-5)M or with the PKA inhibitor, H-89, at 10(-5)M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3 x 10(-6)M or with the PKC inhibitor, GF109203X, at 10(-6)M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC(1)R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/IP(3)/PKC-signaling pathways in goldfish pituitary cells.