Changes in Behavior and Diel Melatonin Secretion Toward Estivation in Western Sand Lance, Ammodytes japonicus (Uranoscopiformes, Ammodytidae).

Western sand lance, Ammodytes japonicus, is known to have an estivation period, in which they cease feeding and stay in the sand from early summer to late autumn, followed by gonadal maturation. During the feeding period prior to estivation, they swim in daytime and spend the night in the sand. Before they start swimming, they show a typical behavior of head-exposing from the sand, which is likely to be related to foraging and predation avoidance. Our previous study revealed that melatonin regulates such diel behavior of this species. To elucidate the mechanisms of behavioral regulation throughout the life cycle of this sand lance, the present study examined the changes in behavior and melatonin secretion toward the estivation period. Both head-exposing and swimming behaviors were frequently observed at the transition period toward estivation. On the other hand, occurrence of these behaviors was suppressed just before entering estivation. Subsequently, it was found that plasma melatonin concentration was about three times higher at night than in daytime in the non-estivation period, while it was retained at high levels throughout the day in the estivation period. These results indicate that diurnal swimming behavior of sand lance from the feeding to estivation periods is associated with the daily cycle of melatonin secretion.

Distribution of pufferfish saxitoxin- and tetrodotoxin-binding protein homolog and tetrodotoxin in the brain and pituitary of juvenile tiger puffer Takifugu rubripes.

Pufferfish saxitoxin- and tetrodotoxin (TTX)-binding protein (PSTBP) is considered to transfer TTX between tissues. The immunohistochemical distribution of PSTBP-homolog (PSTBPh) and TTX in the brain and pituitary of hatchery-reared juvenile tiger puffer Takifugu rubripes was investigated. PSTBPh was observed mainly in the pars intermedia of the pituitary. TTX was only detected in a TTX-fed fish in the neurohypophysis of the pituitary and in several other brain regions. The relationship between PSTBPh and TTX is discussed.

Circadian Rhythm and Endocrinological Control on the Swimming and Sand Burrowing Behaviors of Japanese Sand Lances Ammodytes spp. (Uranoscopiformes, Ammodytidae).

In diurnal and nocturnal organisms, daily activity is regulated by the perception of environmental stimuli and circadian rhythms, which enable organisms to maintain their essential behaviors. The Japanese sand lances genus Ammodytes are coastal marine fish that exhibit unique nocturnal sand burrowing behavior. To elucidate the extrinsic and intrinsic regulation of this behavior and its endocrinological basis, we conducted a series of rearing experiments under various light conditions and hormone administrations. Under a light-dark photoperiod, the fish showed three types of behavior: sand buried, head-exposed from sand, and swimming/feeding. During the transition from dark to light periods, the fish first showed head exposure, followed by swimming and foraging, and buried themselves in the sand immediately after shifting to the dark period. Under constant light conditions, fish exhibited swimming behavior during the period corresponding to the acclimated light period. In addition, swimming did not occur under constant dark conditions but head exposure was observed at the time of the dark-light transition during acclimation. These observations indicate that the essential behavior of sand lances is regulated by both light and circadian rhythms. Subsequently, a melatonin-containing diet promoted the onset of burrowing in 10 to 120 min in a dose-dependent manner at 0.3-128 µg/g-diet, suggesting the direct behavioral regulation by this hormone. These findings suggest that the behavior of sand lances is strictly regulated by an intrinsic mechanism and that melatonin is a regulatory endocrine factor that induces burrowing behavior.

Early Filial Cannibalism in Fish Revisited: Endocrinological Constraint, Costs of Parental Care, and Mating Possibility.

AbstractOffspring desertion by parents generally occurs at an early stage of parental care, which is thought to minimize the costs of parental care prior to desertion. This study investigated the effects of endocrinological constraints on early total filial cannibalism by male Rhabdoblennius nitidus in the field, a paternal brooding blennid fish with androgen-dependent brood cycling. In brood reduction experiments, cannibal males showed low levels of plasma 11-ketotestosterone (11-KT) relative to noncannibals and also similar levels of 11-KT to males in the parental care phase. Since 11-KT regulates male courtship intensity, males with decreased courtship activity would exhibit total filial cannibalism. However, there is a possibility that a transient increase in 11-KT levels at the early stage of parental care delays total filial cannibalism. In contrast, total filial cannibalism could occur before a decline to the lowest 11-KT levels, at which point males might still be able to exhibit courtships, possibly to reduce the costs of parental care. To understand how much and when caregiving males exhibit mating and parental care behaviors, it is important to consider not only the presence of endocrinological constraints but also its intensity and flexibility.

Neuronal responses of melanin-concentrating hormone and corticotropin-releasing hormone to background color in the self-fertilizing fish, Kryptolebias marmoratus.

We examined neuronal responses of hypothalamic melanin-concentrating hormone (MCH) and corticotropin-releasing hormone (CRH) to background color in the self-fertilizing fish, Kryptolebias marmoratus. Fish were individually reared in lidless white or black cylindrical plastic containers for 15 days. The number of MCH-immunoreactive (ir) cell bodies in the nucleus lateralis tuberis (NLT) of the hypothalamus was significantly greater in the white-acclimated fish, while no significant differences were observed in the nucleus anterior tuberis (NAT) of the hypothalamus. Significant differences were not seen in the number of CRH-ir cell bodies in the NLT between the groups. The body of the white- and black-acclimated fish appeared lighter and darker, respectively, compared with the baseline color. In the black-acclimated fish, feeding activity was significantly greater with a tendency toward higher specific growth rate compared with the observations in white-acclimated fish. No significant inter-group cortisol level differences were observed. These results indicate that background color affects MCH neuronal activity in the NLT as well as body color adaptation but does not affect CRH neuronal activity in K. marmoratus.

Taisho-Sanshoku koi have hardly faded skin and show attenuated melanophore sensitivity to adrenaline and melanin-concentrating hormone.

Introduction: Koi carp, an ornamental fish derived from the common carp Cyprinus carpio (CC), is characterized by beautiful skin color patterns. However, the mechanism that gives rise to the characteristic vivid skin coloration of koi carp has not been clarified. The skin coloration of many teleosts changes in response to differences in the background color. This change in skin coloration is caused by diffusion or aggregation of pigment granules in chromatophores and is regulated mainly by sympathetic nerves and hormones. We hypothesized that there would be some abnormality in the mechanism of skin color regulation in koi carp, which impairs skin color fading in response to background color. Methods: We compared the function of melanin-concentrating hormone (MCH), noradrenaline, and adrenaline in CC and Taisho-Sanshoku (TS), a variety of tri-colored koi. Results and Discussion: In CC acclimated to a white background, the skin color became paler and pigment granules aggregated in melanophores in the scales compared to that in black-acclimated CC. There were no clear differences in skin color or pigment granule aggregation in white- or black-acclimated TS. The expression of mch1 mRNA in the brain was higher in the white-acclimated CC than that in the black-acclimated CC. However, the expression of mch1 mRNA in the brain in the TS did not change in response to the background color. Additionally, plasma MCH levels did not differ between white- and black-acclimated fish in either CC or TS. In vitro experiments showed that noradrenaline induced pigment aggregation in scale melanophores in both CC and TS, whereas adrenaline induced pigment aggregation in the CC but not in the TS. In vitro administration of MCH induced pigment granule aggregation in the CC but not in the TS. However, intraperitoneal injection of MCH resulted in pigment granule aggregation in both CC and TS. Collectively, these results suggest that the weak sensitivity of scale melanophores to MCH and adrenaline might be responsible for the lack of skin color change in response to background color in the TS.

Effects of crowding stress on the hypothalamo-pituitary-interrenal axis of the self-fertilizing fish, Kryptolebias marmoratus.

We tested whether crowding stress affects the hypothalamo-pituitary-interrenal (HPI) axis of the self-fertilizing fish, Kryptolebias marmoratus, which is known to be aggressive in the laboratory conditions but sometimes found as a group from a single land crab burrow in the wild. The projection of corticotropin-releasing hormone (CRH) neurons to the adrenocorticotropic hormone (ACTH) cells in the pituitary was confirmed by dual-label immunohistochemistry; CRH-immunoreactive (ir) fibers originating from cell bodies located in the lateral tuberal nucleus (NLT) of the hypothalamus were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Then, fish were reared solitary or in pairs for 14 days, and the number of CRH-ir cell bodies in the NLT of the hypothalamus and cortisol levels in the body without head region were compared. The number of CRH-ir cell bodies and cortisol levels were significantly higher in paired fish. These results indicate that crowding stress affects the HPI axis in K. marmoratus which thrive in small burrows with limited water volume.

Immunohistochemical Localization of a GnRH-Like Peptide in the Nerve Ganglion of Three Classes of Crustaceans, the Tadpole Shrimp Triops longicaudatus (Branchiopoda), the Barnacle Balanus crenatus (Hexanauplia), and the Hermit Crab Pagurus filholi (Malacostraca).

In vertebrates, gonadotropin-releasing hormone (GnRH) regulates gonadal maturation by stimulating the synthesis and release of pituitary gonadotropins. GnRH has also been identified in invertebrates. Crustacea consists of several classes including Cephalocarida, Remipedia, Branchiopoda (e.g., tadpole shrimp), Hexanauplia (e.g., barnacle) and Malacostraca (e.g., shrimp, crab). In the malacostracan crustaceans, the presence of GnRH has been detected in several species, mainly by immunohistochemistry. In the present study, we examined whether a GnRH-like peptide exists in the brain and/or nerve ganglion of three classes of crustaceans, the tadpole shrimp Triops longicaudatus (Branchiopoda), the barnacle Balanus crenatus (Hexanauplia), and the hermit crab Pagurus filholi (Malacostraca), by immunohistochemistry using a rabbit polyclonal antibody raised against chicken GnRH-II (GnRH2). This antibody was found to recognize the giant freshwater prawn Macrobrachium rosenbergii GnRH (MroGnRH). In the tadpole shrimp, GnRH-like-immunoreactive (ir) cell bodies were located in the circumesophageal connective of the deuterocerebrum, and GnRH-like-ir fibers were detected also in the ventral nerve cord. In the barnacle, GnRH-like-ir cell bodies and fibers were located in the supraesophageal ganglion (brain), the subesophageal ganglion, and the circumesophageal connective. In the hermit crab, GnRH-like-ir cell bodies were detected in the anterior-most part of the supraesophageal ganglion and the subesophageal ganglion. GnRH-like-ir fibers were observed also in the thoracic ganglion and the eyestalk. These results suggest that a GnRH-like peptide exists widely in crustacean species.

Effect of the administration of prolactin-releasing peptide2 on feeding activity in the intertidal blenny Rhabdoblennius nitidus (Günther, 1861).

Prolactin-releasing peptide2 (PrRP2) was administered intraperitoneally to male intertidal blenny Rhabdoblennius nitidus, a species with male uniparental care of eggs, to investigate the effect on their feeding activity. A significant inhibitory effect on appetite was observed in the breeding season, but not in the nonbreeding season. These results suggest that PrRP2 and PrRP2 receptors are more active during the breeding season. The presence of a mechanism to inhibit feeding activity while parents take care of their offspring may be important for the success of parental care.

Tetrodotoxin functions as a stress relieving substance in juvenile tiger puffer Takifugu rubripes.

We tested whether tetrodotoxin (TTX) functions as a stress relieving substance in puffer fish. We orally administered TTX to the juveniles of hatchery-reared non-toxic tiger puffer Takifugu rubripes and measured the effects of TTX on brain corticotropin-releasing hormone (CRH) mRNA expression and plasma cortisol levels in comparison with effects in non-toxic juveniles. Firstly, the reciprocal connections of CRH and adrenocorticotropic hormone (ACTH) were confirmed by dual-label immunohistochemistry. CRH-immunoreactive (ir) cell bodies were detected in the hypothalamus and CRH-ir fibers were observed to project to ACTH-ir cells in the rostral pars distalis of the pituitary. Next, a TTX-containing diet (2.35 mouse units (517 ng)/g diet) or a non-toxic diet were fed to the fish for 28 days under a recirculating system. Standard length and body weight became significantly larger in the TTX-treated group. The degree of loss of the caudal fin, which is an indicator of the degree of agonistic interactions, where high values show a higher loss of caudal fin of a fish due to nipping by other individuals, was significantly lower in the TTX-treated group. Relative CRH mRNA expression levels in the brain and cortisol levels in the plasma were significantly lower in the TTX-treated group. These results indicate that TTX functions as a stress relieving substance by affecting the CRH-ACTH-cortisol axis and reducing agonistic interactions in tiger puffer juveniles.

Localization of three forms of gonadotropin-releasing hormone in the brain and pituitary of the self-fertilizing fish, Kryptolebias marmoratus.

The localization of gonadotropin-releasing hormone (GnRH) in the brain and pituitary of the self-fertilizing mangrove killifish Kryptolebias marmoratus was examined by immunohistochemistry and in situ hybridization to understand its neuroendocrine system. The genome assembly of K. marmoratus did not have any sequence encoding GnRH1, but sequences encoding GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH) were found. Therefore, GnRH1 was identified by in silico cloning. The deduced amino acid sequence of the K. marmoratus GnRH1 (mature peptide) was identical to that of the medaka GnRH. GnRH1 neurons were detected in the ventral part of the preoptic nucleus by immunohistochemistry and in situ hybridization, and GnRH1-immunoreactive (ir) fibers were observed throughout the brain. GnRH1-ir fibers were in close contact with luteinizing hormone (LH)-ir cells in the pituitary using double immunohistochemistry. GnRH2 neurons were detected in the midbrain tegmentum by immunohistochemistry and in situ hybridization. Although GnRH2-ir fibers were observed throughout the brain, they were not detected in the pituitary. GnRH3 neurons were detected in the lateral part of the ventral telencephalic area by both methods. GnRH3-ir fibers were observed throughout the brain, and a few GnRH3-ir fibers were in close contact with LH-ir cells in the pituitary. These results indicate that GnRH1 and possibly GnRH3 are responsible for gonadal maturation through LH secretion and that all three forms of GnRH function as neurotransmitters or neuromodulators in the brain of K. marmoratus.

Immunohistochemical localization of GnRH-immunoreactive cell bodies and fibers in the nerve ganglion of Perinereis aibuhitensis (Annelida: Polychaeta).

The gonadotropin-releasing hormone (GnRH) gene sequence has been identified in an annelid polychaete marine worm using continual genome sequencing. The distribution of GnRH immunoreactive (ir) cell bodies and fibers in the nerve ganglion of the clam worm Perinereis aibuhitensis (Polychaeta) was examined by immunohistochemistry using a newly produced rabbit polyclonal antibody raised against the marine worm GnRH (mwGnRH). The specificity of the antibody was confirmed by dot blot assay. The antibody cross-reacted with mwGnRH, but not with other forms of GnRH such as octopus GnRH, tunicate GnRH-I, II, owl limpet GnRH, and lamprey GnRH-II. In P. aibuhitensis, mwGnRH-ir cell bodies were detected in the nuclei 15-22, the caudal part of the cerebral ganglion. Furthermore, mwGnRH-ir fibers were mainly observed in the optic neuropil, but mwGnRH-ir fibers were also detected in the central neuropil region, the subpharyngeal ganglion, and the ventral nerve cord. These results indicate that mwGnRH is synthesized in the cerebral ganglion, is transported through the subpharyngeal ganglion and the ventral nerve cord, and functions either as a neurotransmitter or neuromodulator.

Immunohistochemical detection of prolactin-releasing peptide2 in the brain of the inshore hagfish Eptatretus burgeri.

Prolactin-releasing peptide2 (PrRP2) belongs to the RFamide peptide group and is a paralog of prolactin-releasing peptide (PrRP). Recent studies demonstrated that PrRP2, but not PrRP, regulates prolactin release in teleosts. The evolutionary origin of PrRP and PrRP2 dates back to at least early vertebrates because homologs of PrRP/PrRP2 were identified in lampreys, one of the earliest branch of vertebrates class Agnatha. However, PrRP/PrRP2 remains to be identified in hagfish, another representative species of class Agnatha. Here, we examined the distribution of PrRP2 in the brain and pituitary of the inshore hagfish Eptatretus burgeri to obtain further understanding of the neuroendocrine system of PrRP2. PrRP2-immunoreactive (ir) cell bodies were detected in the infundibular nucleus of hypothalamus (HYinf). PrRP2-ir fibers were restricted around PrRP2-ir cell bodies and were not detected in the dorsal wall of the neurohypophysis compared to the abundant PrRP2-ir fiber distribution in the brain and innervation to the pituitary in other vertebrates. To examine possible reciprocal connections of PrRP2 and other neuropeptides, we further conducted dual-label immunohistochemistry of PrRP2 and the PQRFamide (PQRFa) peptide or corticotropin-releasing hormone (CRH). Reciprocal connections are suggested between PrRP2 and PQRFa neurons as well as between PrRP2 and CRH neurons. The present study demonstrates, for the first time, that PrRP2 is expressed in the brain of inshore hagfish. The restricted distribution of PrRP2-ir fibers in the HYinf suggests that PrRP2 does not directly regulate the pituitary gland, but regulates the function of the HYinf where PQRFa and CRH are expressed.

Immunohistochemical detection of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish, Eptatretus burgeri.

The distribution of corticotropin-releasing hormone (CRH) in the brain and pituitary of the hagfish Eptatretus burgeri, representing the earliest branch of vertebrates, was examined by immunohistochemistry to better understand the neuroendocrine system of hagfish. CRH-immunoreactive (ir) cell bodies were detected in the preoptic nucleus, periventricular preoptic nucleus, infundibular nucleus of the hypothalamus, and in the nucleus "A" of Kusunoki et al. (1982) in the medulla oblongata. In the brain, CRH-ir fibers were detected in almost all areas except for the olfactory bulb and telencephalon. Bundles of CRH-ir fibers were detected in the dorsal wall of the neurohypophysis. However, CRH-ir fibers were distant from adrenocorticotropic hormone (ACTH) cells in the adenohypophysis, as studied by dual-label immunohistochemistry. Cortisol and corticosterone were detected in the plasma by a combination of reverse-phase high performance liquid chromatography and a time-resolved fluoroimmunoassay. These results suggest that in the hagfish, CRH, ACTH, and corticosteroids exist and that CRH released in the neurohypophysis likely reaches the adenohypophysis via diffusion.

Cloning of corticotropin-releasing hormone (CRH) precursor cDNA and immunohistochemical detection of CRH peptide in the brain of the Japanese eel, paying special attention to gonadotropin-releasing hormone.

The stress-related corticotropin-releasing hormone (CRH) was first identified by isolation of its cDNA from the brain of the Japanese eel Anguilla japonica. CRH cDNA encodes a signal peptide, a cryptic peptide and CRH (41 amino acids). The sequence homology to mammalian CRH is high. Next, the distribution of CRH-immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined by immunohistochemistry. CRH-ir cell bodies were detected in several brain regions, e.g., nucleus preopticus pars magnocellularis, nucleus preopticus pars gigantocellularis and formatio reticularis superius. In the brain, CRH-ir fibers were distributed not only in the hypothalamus but also in various regions. Some CRH-ir fibers projected to adrenocorticotropic hormone (ACTH) cells in the rostral pars distalis of the pituitary and also the α-melanocyte-stimulating hormone (α-MSH) cells in the pars intermedia of the pituitary. Finally, the neuroanatomical relationship between the CRH neurons and gonadotropin-releasing hormone (GnRH) neurons was examined by dual-label immunohistochemistry. CRH-ir fibers were found to be in close contact with GnRH-ir cell bodies in the hypothalamus and in the midbrain tegmentum and GnRH-ir fibers were in close contact with CRH-ir cell bodies in the nucleus preopticus pars magnocellularis. These results suggest that CRH has some physiological functions other than the stimulation of ACTH and α-MSH secretion and that reciprocal connections may exist between the CRH neurons and GnRH neurons in the brain of the Japanese eel.

Determination of Ghrelin Structure in the Barfin Flounder (Verasper moseri) and Involvement of Ingested Fatty Acids in Ghrelin Acylation.

Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.

The saccus vasculosus of fish is a sensor of seasonal changes in day length.

The pars tuberalis of the pituitary gland is the regulatory hub for seasonal reproduction in birds and mammals. Although fish also exhibit robust seasonal responses, they do not possess an anatomically distinct pars tuberalis. Here we report that the saccus vasculosus of fish is a seasonal sensor. We observe expression of key genes regulating seasonal reproduction and rhodopsin family genes in the saccus vasculosus of masu salmon. Immunohistochemical studies demonstrate that all of these genes are expressed in the coronet cells of the saccus vasculosus, suggesting the existence of a photoperiodic signalling pathway from light input to neuroendocrine output. In addition, isolated saccus vasculosus has the capacity to respond to photoperiodic signals, and its removal abolishes photoperiodic response of the gonad. Although the physiological role of the saccus vasculosus has been a mystery for several centuries, our findings indicate that the saccus vasculosus acts as a sensor of seasonal changes in day length in fish.

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.

Food deprivation increases the expression of the prepro-orexin gene in the hypothalamus of the barfin flounder, Verasper moseri.

Orexins (orexin-A and -B) are involved in the regulation of food intake in mammals. In the barfin flounder, Verasper moseri, we previously reported that orexin-A-like-immunoreactive (ir) cell bodies are localized in the hypothalamus, which is a possible orexigenic center in fish. However, the physiological roles of orexin in the barfin flounder remain unclear. Here, we cloned prepro-orexin cDNA and examined the effects of feeding status on orexin gene expression in the barfin flounder to obtain a better insight into the roles of orexins in feeding regulation. A molecular cloning study showed that barfin flounder prepro-orexin cDNA encodes a 145 amino acid (aa) polypeptide containing orexin-A (43 aa) and orexin-B (28 aa). Prepro-orexin gene transcripts were detected in the hypothalamus, pituitary, and several peripheral organs such as the eyeball, gills, head kidney, body kidney, spleen, testis, and the skin on the eye-side of the flounder's body. Furthermore, the mean prepro-orexin mRNA expression level in the hypothalamus was significantly higher in fasted than in fed fish. These results show that fasting regulates orexin mRNA in the hypothalamus and suggest that orexin is involved in feeding regulation in barfin flounder.

Anatomical relations between neuropeptide Y, galanin, and gonadotropin-releasing hormone in the brain of chondrostean, the Siberian sturgeon Acipenser baeri.

We investigated the immunohistochemical localization of immunoreactive (ir) cell bodies and fibers of neuropeptide Y (NPY) and galanin (GAL), and the anatomical relations between these neurons in the brain of the Siberian sturgeon Acipenser baeri to clarify the interactions between these neuropeptides. Furthermore, the anatomic relations between NPY and gonadotropin-releasing hormone (GnRH) in the brain were also examined. NPY-ir cell bodies were observed in the ventral part of the ventral telencephalon (Vv). NPY-ir fibers were observed throughout the brain, primarily in the ventral telencephalon, hypothalamus, optic tectum, and midbrain. GAL-ir cell bodies were observed in the Vv, nucleus anterioris tuberis (NAT), nucleus lateralis tuberis (NLT), and nucleus recessus posterioris (NRP). GAL-ir fibers were also observed throughout the brain. Neither NPY-ir fibers nor GAL-ir fibers were detected in the pituitary. Dual-label immunohistochemistry revealed that some GAL-ir fibers were in close contact with NPY-ir cell bodies in the Vv, and some NPY-ir fibers were in close contact with GAL-ir cell bodies in the NAT. Furthermore, some NPY-ir fibers were in close contact with GnRH-ir cell bodies in the preoptic area, and some GnRH-ir fibers were in close contact with NPY-ir cell bodies in the Vv. These findings suggest that reciprocal connections exist between the NPY and GAL neurons and between the NPY and GnRH neurons in the brain of the Siberian sturgeon.