Intraperitoneal administration of arginine vasotocin (AVT) induces anorexigenic and anxiogenic actions via the brain V1a receptor-signaling pathway in the tiger puffer, Takifugu rubripes.

Arginine vasotocin (AVT) is produced mainly in the hypothalamus and as a neurohypophyseal hormone peripherally regulates water-mineral balance in sub-mammals. In addition, AVT-containing neurons innervate several areas of the brain, and AVT also acts centrally as both an anorexigenic and anxiogenic factor in goldfish. However, it is unclear whether these central effects operate in fish in general. In the present study, therefore, we investigated AVT-like immunoreactivity in the brain of the tiger puffer, a cultured fish with a high market value in Japan and also a representative marine teleost species, focusing particularly on whether AVT affects food intake and psychomotor activity. AVT-like immunoreactivity was distributed higher in the ventral region of the telencephalon, the hypothalamus and midbrain. Intraperitoneal (IP) administration of AVT at 100 pmol g-1 body weight (BW) increased the immunoreactivity of phosphorylated ribosomal proteinS6 (RPS6), a neuronal activation marker, in the telencephalon and diencephalon, decreased food consumption and enhanced thigmotaxis. AVT-induced anorexigenic and anxiogenic actions were blocked by IP co-injection of a V1a receptor (V1aR) antagonist, Manning compound (MC) at 300 pmol g-1 BW. These results suggest that AVT acts as an anorexigenic and anxiogenic factor via the V1aR-signaling pathway in the tiger puffer brain.

Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Distribution of neuromedin U (NMU)-like immunoreactivity in the goldfish brain, and effect of intracerebroventricular administration of NMU on emotional behavior in goldfish.

Neuromedin U (NMU) is a multifunctional neuropeptide implicated in regulation of smooth muscle contraction in the circulatory and digestive systems, energy homeostasis and the stress response, but especially food intake in vertebrates. Recent studies have indicated the possible involvement of NMU in the regulation of psychomotor activity in rodents. We have identified four cDNAs encoding three putative NMU variants (NMU-21, -25 and -38) from the goldfish brain and intestine. Recently, we have also purified these NMUs and the truncated C-terminal form NMU-9 from these tissues, and demonstrated their anorexigenic action in goldfish. However, there is no information on the brain localization of NMU-like immunoreactivity and the psychophysiological roles of NMU in fish. Here, we investigated the brain distribution of NMU-like immunoreactivity and found that it was localized throughout the fore- and mid-brains. We subsequently examined the effect of intracerebroventricular (ICV) administration of NMU-21, which is abundant only in the brain on psychomotor activity in goldfish. As goldfish prefer the lower to the upper area of a tank, we developed an upper/lower area preference test in a tank for evaluating the psychomotor activity of goldfish using a personal tablet device without an automatic behavior-tracking device. ICV administration of NMU-21 at 10 pmol g-1 body weight (BW) prolonged the time spent in the upper area of the tank, and this action mimicked that of ICV administration of the central-type benzodiazepine receptor (CBR) agonist tofisopam at 100 pmol g-1 BW. These results suggest that NMU-21 potently induces anxiolytic-like action in the goldfish brain.

High-sucrose diets contribute to brain angiopathy with impaired glucose uptake and psychosis-related higher brain dysfunctions in mice.

Metabolic dysfunction is thought to contribute to the severity of psychiatric disorders; however, it has been unclear whether current high­simple sugar diets contribute to pathogenesis of these diseases. Here, we demonstrate that a high-sucrose diet during adolescence induces psychosis-related behavioral endophenotypes, including hyperactivity, poor working memory, impaired sensory gating, and disrupted interneuron function in mice deficient for glyoxalase-1 (GLO1), an enzyme involved in detoxification of sucrose metabolites. Furthermore, the high-sucrose diet induced microcapillary impairments and reduced brain glucose uptake in brains of Glo1-deficient mice. Aspirin protected against this angiopathy, enhancing brain glucose uptake and preventing abnormal behavioral phenotypes. Similar vascular damage to our model mice was found in the brains of randomly collected schizophrenia and bipolar disorder patients, suggesting that psychiatric disorders are associated with angiopathy in the brain caused by various environmental stresses, including metabolic stress.

Intracerebroventricular administration of α-melanocyte-stimulating hormone (α-MSH) enhances thigmotaxis and induces anxiety-like behavior in the goldfish Carassius auratus.

α-Melanocyte-stimulating hormone (α-MSH) is a body pigmentation-regulating hormone secreted from the intermediate lobe of the pituitary in vertebrates. It is also produced in the brain, and acts as an anorexigenic neuropeptide involved in feeding regulation. In rodents, intracerebroventricular (ICV) administration of α-MSH has been shown to affect not only feeding behavior, but also psychomotor activity. However, there is still no information regarding the psychophysiological effects of α-MSH on behavior in fish. Therefore, we examined the effect of synthetic α-MSH on psychomotor activity in goldfish. Since this species prefers the edge to the central area of a tank, we used this as a preference test for assessing psychomotor activity. When α-MSH was administered ICV at 1 and 10 pmol g-1 body weight (BW), the time spent in the edge area of a tank was prolonged at 10 pmol g-1 BW. However, α-MSH at these doses did not affect locomotor activity. The action of α-MSH mimicked those of FG-7142 (a central-type benzodiazepine receptor (CBR) inverse agonist with an anxiogenic effect) at 10 pmol g-1 BW and melanotan II (a melanocortin 4 receptor (MC4R) agonist) at 50 pmol g-1 BW, whereas ICV administration of tofisopam (a CBR agonist with an anxiolytic effect) at 10 pmol g-1 BW prolonged the time spent in the central area. The anxiogenic-like effect of α-MSH was abolished by treatment with the MC4R antagonist HS024 at 50 pmol g-1 BW. These data indicate that α-MSH affects psychomotor activity in goldfish, and exerts an anxiogenic-like effect via the MC4R-signaling pathway.

Identification and signaling characterization of four urotensin II receptor subtypes in the western clawed frog, Xenopus tropicalis.

Urotensin II (UII) is involved, via the UII receptor (UTR), in many physiological and pathological processes, including vasoconstriction, locomotion, osmoregulation, immune response, and metabolic syndrome. In silico studies have revealed the presence of four or five distinct UTR (UTR1-UTR5) gene sequences in nonmammalian vertebrates. However, the functionality of these receptor subtypes and their associations to signaling pathways are unclear. In this study, full-length cDNAs encoding four distinct UTR subtypes (UTR1, UTR3, UTR4, and UTR5) were isolated from the western clawed frog (Xenopus tropicalis). In functional analyses, homologous Xenopus UII stimulation of cells expressing UTR1 or UTR5 induced intracellular calcoum mobilization and phosphorylation of extracellular signal-regulated kinase 1/2. Cells expressing UTR3 or UTR4 did not show this response. Furthermore, UII induced the phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) through the UII-UTR1/5 system. However, intracellular cAMP accumulation was not observed, suggesting that UII-induced CREB phosphorylation is caused by a signaling pathway different from that involving Gs protein. In contrast, the administration of UII to cells increased the phosphorylation of guanine nucleotide exchange factor-H1 (GEF-H1) and myosin light chain 2 (MLC2) in all UTR subtypes. These results define four distinct UTR functional subtypes and are consistent with the molecular evolution of UTR subtypes in vertebrates. Further understanding of signaling properties associated with UTR subtypes may help in clarifying the functional roles associated with UII-UTR interactions in nonmammalian vertebrates.

Molecular Identification, Characterization, and Expression Analysis of a Metallothionein Gene from Septifer virgatus.

This study provides a preliminary characterization of a metallothionein (MT) gene in Septifer virgatus and highlights its potential use in biomonitoring. The full-length SvMT cDNA and the complete sequence of the SvMT gene were identified using reverse transcriptase PCR coupled with the rapid amplification of cDNA ends and the primer walking method. The SvMT cDNA encodes a protein of 72 amino acids having nine classical Cys-X-Cys motifs. Moreover, the deduced amino acids contained the conserved motif (Cys-x-Cys-x(3)-Cys-Thr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys) of MT family 2. Its molecular mass and isoelectric point were estimated to be 7.01 kDa and 7.00, respectively. BLAST-based searching indicated that SvMT shared 81.0% amino acid sequence identity with Mytilus edulis MT-20-II. The SvMT gene has three coding exons and two introns. After exposure to 1 mg/L cadmium chloride, the expression of SvMT increased 15-fold by 3 days (d), with a maximum expression of 27-fold by 5 d compared with the pre-exposure level. After exposure to 2 mg/L zinc chloride, the expression of SvMT increased 2.5-fold by 3 d and 4.7-fold by 5 d compared with the pre-exposure level. A significant increase in the expression level of SvMT mRNA was observed after the exposure of S. virgatus to the combination of 0.003 mg/L cadmium chloride and 0.2 mg/L zinc chloride compared with the pre-exposure level. Our work indicates that the SvMT gene is associated with stress responses and could be a potential biomarker for marine pollution.

Effect of intracerebroventricular administration of two molecular forms of sulfated CCK octapeptide on anxiety-like behavior in the zebrafish danio rerio.

Cholecystokinin octapeptide with sulfate (CCK-8s) regulates feeding behavior and psychomotor activity. In rodents and goldfish, intracerebroventricular (ICV) injection of CCK-8s decreases food intake and also induces anxiety-like behavior. The zebrafish has several merits for investigating the psychophysiological roles of neuropeptides. However, little is known about the brain localization of CCK and the behavioral action of CCK-8s in this species. Here we investigated the brain localization of CCK-like immunoreactivity and found that it was distributed throughout the brain. As CCK-like immunoreactivity was particularly evident in the ventral habenular nucleus, the interpeduncular nucleus and superior raphe, we subsequently examined the effect of zebrafish (zf) CCK-8s on psychomotor control. Since the zebrafish possesses two molecular forms of zfCCK-8s (zfCCKA-8s and zfCCKB-8s), two synthetic peptides were administered intracerebroventricularly at 1, 5 and 10 pmol g-1 body weight (BW). As the zebrafish shows a greater preference for the lower area of a tank than for to the upper area, we used this preference for assessment of anxiety-like behavior. ICV administration of zfCCKA-8 s or zfCCKB-8s at 10 pmol g-1 BW significantly shortened the time spent in the upper area. The actions of these peptides mimicked that of the central-type benzodiazepine receptor inverse agonist FG-7142 (an anxiogenic agent) at 10 pmol g-1 BW. The anxiogenic-like action of the two peptides was attenuated by treatment with the CCK receptor antagonist proglumide at 200 pmol g-1 BW. These results indicate that zfCCKA-8s and zfCCKB-8s potently induce anxiety-like behavior via the CCK receptor-signaling pathway in the zebrafish brain.

Lipopolysaccharide (LPS) inhibits ectopic bone formation induced by bone morphogenetic protein-2 and TGF-ß1 through IL-1ß production.

OBJECTIVES: In order to gain new insight into bacterial infection during bone-regenerative treatment using bone morphogenetic proteins (BMPs), we examined the effects of lipopolysaccharide (LPS) on ectopic bone formation induced by BMP-2 and transforming growth factor (TGF)-ß1 in mice. METHODS: We implanted collagen sponges containing BMP-2, TGF-ß1, and various amounts of LPS into mouse muscle tissues. Lump-like masses in which ectopic bones developed in mice were processed for microcomputed tomography, DNA microarray, reverse-transcription PCR, and histological analyses. RESULTS: LPS treatment caused a dose-dependent reduction in the volume of ectopic bone. The total volume of ectopic bone induced by BMP-2 + TGF-ß1 treatment was reduced by more than 75% in the presence of LPS. Histological analysis of the ectopic bone tissues revealed a significant reduction in total bone volume and bone volume/total volume in response to LPS. LPS treatment significantly increased the osteoblast number and osteoid volume, while the osteoclast number did not change. Since LPS induced production of TNF-α and IL-1ß in lump-like masses, we implanted collagen sponges containing BMP-2 and TGF-ß1 with or without LPS into TNF-α- or IL-1α/ß-deficient mice. LPS treatment reduced the volume of ectopic bones in TNF-α-deficient mice but not in IL-1α/ß-deficient mice. Furthermore, collagen sponges containing IL-1ß reduced ectopic bone formation by BMP-2 and TGF-ß1 in wild-type mice to the same extent as LPS treatment did. CONCLUSIONS: LPS suppresses the ectopic bone formation induced by BMP-2 and TGF-ß1 through IL-1ß production.

Pituitary Adenylate Cyclase-Activating Polypeptide in the Ventromedial Hypothalamus Is Responsible for Food Intake Behavior by Modulating the Expression of Agouti-Related Peptide in Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is abundantly expressed in the hypothalamus and contributes to hypothalamic functions, including appetite regulation. Although food intake is suggested to be decreased in PACAP (-/-) mice, the detailed mechanisms are still being discussed. We sought to investigate this link. The food consumption at 8 h after refeeding in the (-/-) mice who had fasted for 2 days was significantly lower than in the PACAP (+/+) mice. The nocturnal and daily food intake of (-/-) mice was significantly lower than those of (+/+) mice, but the diurnal food intake showed a tendency to increase. mRNA expression levels of agouti-related peptide (AgRP) were decreased, but those of proopiomelanocortin (POMC) were increased in the hypothalamus of (-/-) mice 4 h after refeeding. Furthermore, intracerebroventricular administration of a PACAP receptor antagonist, PACAP6-38 (1 nmol/4 µL/mouse), decreased food intake and body weight 1, 2, and 4 h after refeeding, as well as expression levels of AgRP at 4 h after refeeding in (+/+) mice. The selective overexpression of PACAP by the infection of an adeno-associated virus in the ventromedial hypothalamus (VMH) resulted in an increase in food intake and AgRP expression in the nocturnal period in addition to the increased food intake at 8 h after refeeding. These results suggest that food intake behavior in mice is triggered by the increase in PACAP expression in the VMH via modulation of AgRP expression in the hypothalamus, pointing to PACAP inhibition as a potential strategy for the development of anti-obesity drugs.

Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks.

Intracerebroventricular administration of arginine vasotocin (AVT) induces anorexigenesis and anxiety-like behavior in goldfish.

Arginine vasotocin (AVT) is known as a neurohypophyseal hormone that regulates water- and mineral-balance in non-mammalian vertebrates. Recent studies revealed that AVT also exerts central effects on behavior. The goldfish has several merits for evaluation of behavioral changes. However, there is few information on the behavioral action of AVT in this species. Here we examined the effects of AVT on food intake and psychomotor activity. AVT was administered intracerebroventricularly at 1, 5 and 10 pmol g-1 body weight (BW). Intracerebroventricular (ICV) administration of AVT at 5 and 10 pmol g-1 BW significantly decreased food intake during 30 min after injection and recovery from anesthesia. The AVT-induced anorexigenic action was attenuated by treatment with the AVT receptor V1aR antagonist Manning compound (MC) at 50 pmol g-1 BW. As the goldfish tends to prefer the lower to the upper area of a tank, we used this preference behavior for assessing psychomotor activity during a 30-min observation period. ICV administration of AVT at 1, 5 and 10 pmol g-1 BW significantly prolonged the time spent in the lower area, but did not affect locomotor activity in the tank at any dose. The action of AVT was similar to that of the central-type benzodiazepine receptor inverse agonist FG-7142 at 10 pmol g-1 BW. AVT-induced anxiety-like behavior was blocked by treatment with MC at 50 pmol g-1 BW. These results indicate that AVT affects food intake and psychophysiological status, and also induces anorexigenic- and anxiogenic-like actions via the V1aR-signaling pathway in the goldfish brain.

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates.

Effects of PACAP on Dry Eye Symptoms, and Possible Use for Therapeutic Application.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide/glucagon/secretin family of peptides. PACAP and its three receptor subtypes are expressed in neural tissues and in the eye, including the retina, cornea, and lacrimal gland. PACAP is known to exert pleiotropic effects on the central nervous system and in eye tissues where it plays important roles in protecting against dry eye. This review provides an overview of current knowledge regarding dry eye symptoms in aged animals and humans and the protective effects, mechanisms of action. In addition, we also refer to the development of a new preventive/therapeutic method by PACAP of dry eye patients.

Intracerebroventricular administration of sulphated cholecystokinin octapeptide induces anxiety-like behaviour in goldfish.

Sulphated cholecystokinin octapeptide (CCK-8s) is involved in feeding regulation as an anorexigenic neuropeptide in vertebrates. In rodents, i.c.v. administration of CCK-8s has been shown to affect not only feeding behaviour, but also psychomotor activity. However, there is still no information available concerning the psychophysiological effects of CCK-8s in goldfish. Therefore, in the present study, we examined the effect of synthetic goldfish (gf) CCK-8s on psychomotor activity in this species. Intracerebroventricular administration of gfCCK-8s at 0.1, 0.5 and 2.5 pmol g-1 body weight (BW) did not affect swimming distance (locomotor activity). Because goldfish prefer the lower to the upper area of a tank, we used this as a preference test (upper/lower test) to assess anxiety-like behaviour. Intracerebroventricular administration of gfCCK-8s at 2.5 pmol g-1 BW shortened the time spent in the upper area. The action of gfCCK-8s mimicked that of FG-7142 (the central-type benzodiazepine receptor inverse agonist, an anxiogenic agent) at 5 and 10 pmol g-1 BW. The anxiogenic-like effect of gfCCK-8s was abolished by treatment with the CCK receptor antagonist proglumide at 50 pmol g-1 BW. We also investigated the localisation of CCK/gastrin-like immunoreactivity in the goldfish brain. CCK/gastrin-like immunoreactivity was observed in the anxiety-related regions (the nucleus habenularis and the interpeduncular nucleus). These data indicate that gfCCK-8s potently affects psychomotor activity in goldfish, and exerts an anxiogenic-like effect via the CCK receptor-signalling pathway.

[Novel tear secretion system - the effect and the mechanism of PACAP on tear secretion].

Dry eye syndrome is defined as a disorder of the tear film caused by either a decreased production in tears or a disruption to the stability of the complex tear film, which causes damage to the ocular surface. It has been developed the medicine for dry eye syndrome focusing anti-inflammation or mucin secretion, however, no treatment has been developed focusing on the effect of elevation of the lacrimal secretion. We recently identified that pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dry eye-like symptoms such as corneal keratinization and tear reduction. PACAP receptor (PAC1-R) immunoreactivity was observed in the acinar cells of the mouse lacrimal gland. PACAP eye drop significantly stimulated tear secretion level, and the effect was suppressed by pretreatment with PAC1-R antagonist or adenylate cyclase inhibitor. PACAP eye drop on the PACAP KO mouse significantly increased the tear secretion, and continuous eye drop suppressed progression of the corneal keratinization. PACAP eye drops increase aquaporin 5 (AQP5) levels in the membrane of acinar cells in lacrimal glands. AQP5 siRNA treatment significantly attenuates PACAP-induced tear secretion. Based on these results, PACAP might be clinically useful to treat dry eye disorder.

Discovery of PACAP and its receptors in the brain.

Pituitary adenylate-cyclase-activating polypeptide (PACAP) is a 27- or 38-amino acid neuropeptide, which belongs to the vasoactive intestinal polypeptide (VIP)/glucagon/secretin family. PACAP shows particularly high homology (~ 68%) to VIP. Because of the high homology of the amino acid sequences of PACAP and VIP, these peptides share three class B-G-protein coupled receptors: the PAC1-Receptor (PAC1-R), the VPAC1-Receptor (VPAC1-R) and VPAC2-Receptor (VPAC2-R). These receptors have high homology to each other, and their high homology is utilized for these discoveries. This review provides mainly an overview of the history of the discovery of PACAP and its three receptors.

Distribution of pituitary adenylate cyclase-activating polypeptide 2 in zebrafish brain.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multipotent neuropeptide with an amino acid sequence that is well conserved among vertebrates. In teleosts, including zebrafish, the PACAP gene (adcyap1) has been duplicated to yield adcyap1a (coding PACAP1) and adcyap1b (coding PACAP2). This study aims to determine the distribution of these PACAPs and their mRNAs in zebrafish. We generated a zebrafish PACAP2-specific antibody. Using real-time PCR, we observed that adcyap1b mRNA was primarily localized in the brain, with the highest level in the telencephalon, followed by the diencephalon. Using immunostaining of brain tissue samples, PACAP2 immunoreactivity was observed mainly in the telencephalon, hypothalamus, and cerebellum, and the immunopositive fibers formed a line to the habenula. PACAP2-immunopositive cells were observed in the ventral and dorsal regions of the telencephalon and in the hypothalamic nucleus of the diencephalon in the colchicine-injected brain. This distribution of PACAP2 suggests its involvement in higher brain functions in teleosts, such as learning and cognition, as well as instinctive behaviors such as feeding and emotional regulation.

Neuropeptide Y-Induced Orexigenic Action Is Attenuated by the Orexin Receptor Antagonist in Bullfrog Larvae.

In bullfrog larvae at the pre- and pro-metamorphic stages, feeding behavior is regulated by appetite factors such as orexigenic peptides. In fact, food intake is enhanced by intracerebroventricular (ICV) administration of neuropeptide Y (NPY) and orexin A. Using goldfish, our previous study indicated that the orexigenic action of NPY is mediated by orexin A, suggesting the functional interaction between the two. However, there is little information about whether the action of orexin A mediates the orexigenic action of NPY in bullfrog larvae. Therefore, we examined the effect of the orexin receptor antagonist, SB334867 on the orexigenic action of NPY in larvae. The stimulatory effect of ICV injection of NPY at 10 pmol/g body weight (BW) on food intake was abolished by treatment with SB334867 at 60 pmol/g BW. These results suggest that, in bullfrog larvae, there is a neuronal relationship between the NPY and orexin systems, and that the orexigenic action of NPY is mediated by the orexin A-induced orexigenic action.

Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish.

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.