Nucleus Posterioris Periventricularis Research Articles

Response of Neuropeptides to Hunger Signals in Teleost

Introduction: The perception of hunger is a complex physiological process that requires precise coordination between the central and peripheral tissues. Methods: In this study, tilapia fasted for 24 h was chosen to establish a hunger model to study the mechanism of homeostasis recovery under the joint regulation of the central nervous system (CNS) and peripheral tissues. Results: The gastric and intestinal contents of tilapia were predominantly depleted after a fasting period of 9 h and 24 h, respectively. The serum glucose level significantly decreased at the 9-h and 24-h fasting, respectively, and the glucokinase-dependent glucosensing mechanism in the liver was identified as well as the significant activation of phospho-AMPK. However, fasting for 24 h did not activate glucosensing mechanisms and AMPK signaling pathways in the hypothalamus. On the other hand, significant reductions were observed in the mRNA levels of the lipid synthesis-related genes fas and accα, and the serum triglyceride levels as well. The mRNA levels of npy, agrp, pomc, and cart in the hypothalamus fluctuated during the fasting period without significant differences. With in situ hybridization npy signals upregulated in the ventral zone of posterior periventricular nucleus after 24-h fasting, pomc signals enhanced in the lateral tuberal nucleus. Based on the serum metabolomic analysis, the levels of branched-chain amino acids, butyrate, and short-chain acylcarnitine decreased, while those of medium- and long-chain acylcarnitine increased. Conclusion: Fasting for 24 h resulted in changes in npy and pomc signals within the hypothalamus and triggered the glucosensing mechanism in the liver of tilapia. This study is beneficial for elucidating the response of neuropeptides in the CNS to the changes of nutritional factors when hungry.

Characterization and distribution of kisspeptins, kisspeptin receptors, GnIH, and GnRH1 in the brain of the protogynous bluehead wrasse (Thalassoma bifasciatum)

The kisspeptin and gonadotropin-inhibitory hormone (GnIH) systems regulate the hypothalamic-pituitary-gonadal (HPG) axis in a broad range of vertebrates through direct or indirect effects on hypothalamic/preoptic gonadotropin-releasing hormone (GnRH) neurons and pituitary gonadotropes. These systems are sensitive to environmental factors, including social conditions, and may assist in relaying environmental signals to the HPG axis in a potentially broad range of taxa. In this study, we characterized expression of kisspeptin-system genes (kiss1, kiss2, kissr1, and kissr2), gnih, and gnrh1 in the brain of the bluehead wrasse (Thalassoma bifasciatum), an important teleost model of socially-controlled sex change. We analyzed cDNA sequences and examined transcript distributions in the brain using in situ hybridization (ISH) to determine if expression occurs in reproductively-relevant and conserved regions. Expression of kiss1 was detected in the habenula, lateral hypothalamic nucleus (LHn), and preoptic area (POA), while kiss2 was expressed in the dorsal hypothalamus, with sporadic signal in the POA. Expression of kissr1 was detected in the POA, habenula, and LHn, while kissr2 expression was widespread. Gnih mRNA was detected in the posterior periventricular nucleus (NPPv), and gnrh1 neurons localized to the POA. Neurons expressing kissr2 and gnih co-regionalized in the NPPv, while kissr1, kissr2, and gnrh1 co-regionalized in the POA. Double-label ISH revealed very close proximity between kissr1 and gnrh1 neurons, suggesting potential communication between the kisspeptin and GnRH1 systems through these interneurons. These expression patterns are generally conserved and suggest that if kisspeptins do signal GnRH1 neurons, the interaction is indirect, possibly through neurons adjacent to GnRH1. With this foundation in place, future studies can help determine the interactions among these systems and whether these peptides assist in transducing social changes into a shift from female to male sexual function.

Organization of the gonadotropin-inhibitory hormone (Lpxrfa) system in the brain of zebrafish (Danio rerio)

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds and mammals. However, the role of GnIH (Lpxrfa) in teleosts is unknown. In this study, a transgenic zebrafish (Danio rerio) line Tg(gnih:mCherry) was developed to determine the organization of GnIH neurons in the brain. Another transgenic line, Tg(gnih:mCherry; gnrh3:eGFP), was established to determine the positional relationships between GnIH and GnRH3 neurons. In these transgenic lines, the mCherry protein was specifically expressed in GnIH neurons, and eGFP was expressed exclusively in GnRH3 neurons. We found that GnIH cell somata were restricted to the posterior periventricular nucleus (NPPv). Most GnIH neuronal processes projected to the hypothalamus, but a few extended to the posterior tuberculum, telencephalon, and olfactory bulb. GnIH neuronal processes were in close apposition with GnRH3 cell somata and processes in the preoptic-hypothalamic area but were seldom in direct contact. However, in the olfactory bulb, GnIH neuronal processes were in proximity to the terminal nerve GnRH3 cell somata. Neither GnIH cell soma nor neuronal processes were detected in the pituitary, although GnIH receptor mRNAs (npffr1l1, npffr1l2, and npffr1l3) were detected. Intraperitoneal administration of GnIH-3 peptides promoted the transcription of brain gnrh3 as well as pituitary fshβ but not lhβ. Thus, GnIH cell somata were specifically distributed in the NPPv, and their fibers extended to the hypothalamus and advanced to the telencephalon and olfactory bulb. We conclude that GnIH may directly stimulate terminal nerve GnRH3 neurons in the zebrafish brain.

Sexually dimorphic neuronal inputs to the neuroendocrine dopaminergic system governing prolactin release.

Prolactin (PRL) is a pleiotropic hormone that was identified in the context of maternal care and its release from the anterior pituitary is primarily controlled by neuroendocrine dopaminergic (NEDA) neurones of the arcuate nucleus of the hypothalamus. The sexually dimorphic nature of PRL physiology and associated behaviours is evident in mammals, even though the number and density of NEDA neurones is reported as not being sexually dimorphic in rats. However, the underlying circuits controlling NEDA neuronal activity and subsequent PRL release are largely uncharacterised. Thus, we mapped whole‐brain monosynaptic NEDA inputs in male and female mice. Accordingly, we employed a rabies virus based monosynaptic tracing system capable of retrogradely mapping inputs into genetically defined neuronal populations. To gain genetic access to NEDA neurones, we used the dopamine transporter promoter. Here, we unravel 59 brain regions that synapse onto NEDA neurones and reveal that male and female mice, despite monomorphic distribution of NEDA neurones in the arcuate nucleus of the hypothalamus, receive sexually dimorphic amount of inputs from the anterior hypothalamic nucleus, anteroventral periventricular nucleus, medial preoptic nucleus, paraventricular hypothalamic nucleus, posterior periventricular nucleus, supraoptic nucleus, suprachiasmatic nucleus, lateral supramammillary nucleus, tuberal nucleus and periaqueductal grey. Beyond highlighting the importance of considering sex as a biological variable when evaluating connectivity in the brain, these results illustrate a case where a neuronal population with similar anatomical distribution has a subjacent sexually dimorphic connectivity pattern, potentially capable of contributing to the sexually dimorphic nature of PRL release and function.

Ontogeny of Gonadotropin-Inhibitory Hormone (GnIH) in the cichlid fish Cichlasoma dimerus.

RFamide peptides are expressed in the early stages of development in most vertebrates. Gonadotropin-inhibitory hormone (GnIH) belongs to the RFamide family, and its role in reproduction has been widely studied in adult vertebrates, ranging from fish to mammals. As only three reports evaluated GnIH during development, the aim of this study was to characterise the ontogeny of GnIH in a fish model, Cichlasoma dimerus. We detected the presence of two GnIH-immunoreactive (GnIH-ir) cell clusters with spatial and temporal differences. One cluster was observed by 3 days post-hatching (dph) in the nucleus olfacto-retinalis (NOR) and the other in the nucleus posterioris periventricularis by 14 dph. The number of GnIH-ir neurons increased in both nuclei, whereas their size increased only in the NOR from hatchling to juvenile stages. These changes occurred from the moment larvae started feeding exogenously and during development and differentiation of gonadal primordia. We showed by double-label immunofluorescence that only GnIH-ir neurons in the NOR co-expressed GnRH3 associated peptide. In addition, GnIH-ir fibre density increased in all brain regions from 5 dph. GnIH-ir fibres were also detected in the retina, optic tract and optic tectum, suggesting that GnIH acts as a neuromodulator of photoreception and the integration of different sensory modalities. Also, there were GnIH-ir fibres in the pituitary from 14 dph, which were in close association with somatotropes. Moreover, GnIH-ir fibres were observed in the saccus vasculosus from 30 dph, suggesting a potential role of GnIH in the modulation of its function. Finally, we found that gnih was expressed from 1 dph, and that the pattern of variation of its transcript levels was in accordance with that of cell number. Present results are the starting point for the study of new GnIH roles during development. This article is protected by copyright. All rights reserved.

Social rank-dependent expression of gonadotropin-releasing hormones and kisspeptin in the medaka brain

Social interactions regulate the expression of several neuropeptides that have a central role in the reproductive system of mammals. Nonmammalian vertebrates also have these neuropeptides or paralogs, however, studies on the social regulation of reproductive physiology in nonmammalian species are limited. In this study, we examined whether the expression of gonadotropin-releasing hormones (GnRHs) and kisspeptin (Kiss1) is affected by social hierarchy resulting from the outcomes of male-male competition in medaka fish (Oryzias latipes). Four males were introduced to each other in an experimental tank, and classified as the most aggressive dominant or the most submissive subordinate fish, based on the frequency of their aggressive acts during a short-term competition. Dominant and subordinate males maintained their social rank during a long-term competition. Immediately after short-term competition, gonadotropin-releasing hormone-3 (GnRH3) level in the olfactory bulb was significantly higher in subordinate males than in dominant males. After long-term competition, dominant males had high level of gonadotropin-releasing hormone-1 (GnRH1) in the preoptic area, whereas subordinate males had lower Kiss1 level in the nucleus ventral tuberis. On the other hand, the levels of gonadotropin-releasing hormone-2 (GnRH2) in the nucleus lateralis valvulae and Kiss1 in the nucleus posterioris periventricularis, and plasma 11-ketotestosterone (11-KT) concentration did not differ between subordinate and dominant males after the short- and long-term competitions. These results suggest that social hierarchy regulates the expression of GnRH1, GnRH3, and Kiss1 without affecting 11-KT level in male medaka.

Gonadotrophin‐Inhibitory Hormone in the Cichlid Fish Cichlasoma dimerus: Structure, Brain Distribution and Differential Effects on the Secretion of Gonadotrophins and Growth Hormone

The role of gonadotrophin-inhibitory hormone (GnIH) in the inhibition of the reproductive axis has been well-established in birds and mammals. However, its role in other vertebrates, such as the teleost fish, remains controversial. In this context, the present study aimed to evaluate whether GnIH modulates the release of gonadotrophins and growth hormone (GH) in the cichlid fish Cichlasoma dimerus. First, we partially sequenced the precursor polypeptide for GnIH and identified three putative GnIH peptides. Next, we analysed the expression of this precursor polypeptide via a polymerase chain reaction in the reproductive axis of both sexes. We found a high expression of the polypeptide in the hypothalamus and gonads of males. Immunocytochemistry allowed the observation of GnIH-immunoreactive somata in the nucleus posterioris periventricularis and the nucleus olfacto-retinalis, with no differences between the sexes. GnIH-immunoreactive fibres were present in all brain regions, with a high density in the nucleus lateralis tuberis and at both sides of the third ventricle. Finally, we performed invitro studies on intact pituitary cultures to evaluate the effect of two doses (10(-6) m and 10(-8) m) of synthetic C.dimerus (cd-) LPQRFa-1 and LPQRFa-2 on therelease of gonadotrophins and GH. We observed that cd-LPQRFa-1 decreased β-luteinising hormone (LH) and β-follicle-stimulating hormone (FSH) and also increased GH release to the culture medium. The release of β-FSH was increased only when it was stimulated with the higher cd-LPQRFa-2 dose. The results of the present study indicate that cd-LPQRFa-1, the cichlid fish GnIH, inhibits β-LH and β-FSH release and stimulates GH release in intact pituitary cultures of C.dimerus. The results also show that cd-LPQRF-2 could act as an β-FSH-releasing factor in this fish species.

Orexin and neuropeptide Y: Tissue specific expression and immunoreactivity in the hypothalamus and preoptic area of the cichlid fish Cichlasoma dimerus

Neuropeptide Y (NPY) and orexin are neuropeptides involved in the regulation of feeding in vertebrates. In this study we determined the NPY and orexin mRNA tissue expression and their immunoreactivity distribution in both preoptic area and hypothalamus, regions involved in the regulation of feeding behavior. Both peptides presented a wide expression in all tissues examined. The NPY-immunoreactive (ir) cells were localized in the ventral nucleus posterioris periventricularis (NPPv) and numerous ir-NPY fibers were found in the nucleus lateralis tuberis (NLT), the nucleus recess lateralis (NRL) and the neurohypophysis. Ir-orexin cells were observed in the NPPv, dorsal NLT, ventral NLT, lateral NLT (NLTl) and the lateral NRL. Ir-orexin fibers were widespread distributed along all the hypothalamus, especially in the NLTl. Additionally, we observed the presence of ir-orexin immunostaining in adenohypophyseal cells, especially in somatotroph cells and the presence of a few ir-orexin-A fibers in the neurohypophysis. In conclusion, both peptides have an ubiquitous mRNA tissue expression and are similarly distributed in the hypothalamus and preoptic area of Cichlasoma dimerus. The presence of ir-orexin in adenohypohyseal cells and the presence of ir-orexin and NPY fibers in the neurohypophysis suggest that both peptides may play an important neuroendocrine role in anterior pituitary.

Immunohistochemical localization of serotonin in the brain during natural sex change in the hermaphroditic goby Lythrypnus dalli

The neurotransmitter serotonin (5-HT) may play a central role in the inhibition of socially regulated sex change in fish because of its known modulation of both aggressive and reproductive behavior. This is the first study to use immunohistochemical techniques to examine the morphometry of serotonergic neurons at different times during sex change. Using a model species wherein sex change is socially regulated via agonistic social interactions (the bluebanded goby, Lythrypnus dalli), we sampled brains of males and females with different social status, and of females at different times during sex change. Consistent with previous studies on other teleosts, immunoreactive neurons were found in the posterior periventricular nucleus (NPPv), the nucleus of the lateral recess (NRL), the nucleus of the posterior recess (NRP) and in the raphe nucleus. We measured the total area of NPPv, NRL, NRP, and the number and mean cell area of serotonergic neurons in the raphe nucleus. There was no significant difference in any of the brain regions between males, females or sex changing fish, but there was a slight increase in the number of dorsal raphe neurons in the brain of sex changers 2 h after male removal. The results show that in L. dalli the serotonergic system does not present any morphological sex and status differences, nor any dramatic modifications during sex change. These data, together with previous results, do not support the hypothesis that serotonin inhibits socially regulated sex change.

Forebrain mapping of secretoneurin‐like immunoreactivity and its colocalization with isotocin in the preoptic nucleus and pituitary gland of goldfish

Secretoneurin, a 33-34 amino acid neuropeptide derived from the proteolytic processing of the secretogranin-II precursor protein, is reasonably well conserved in evolution. Goldfish secretoneurin shares >75% similarity overall with other vertebrate secretoneurin sequences. The secretoneurin peptide has numerous functions that include neuroinflammation, neurotransmitter release, and neuroendocrine regulation. A detailed description of the central distribution of secretoneurin immunoreactivity is only known for the rat. Using our polyclonal antibody against the central, conserved core of the secretoneurin peptide we studied the distribution of secretoneurin-like immunoreactivity in the goldfish brain. Secretoneurin immunoreactivity was found in the olfactory bulb, entopeduncular nucleus, preoptic nucleus, lateral part of the lateral tuberal nucleus, posterior periventricular nucleus, nucleus of the posterior recess, the nucleus of the saccus vasculosus, and nucleus isthmi. Secretoneurin-immunoreactive fibers were found in the dorsal part of the dorsal telencephalon, ventral and lateral parts of the ventral telencephalon, periventricular preoptic nucleus, pituitary, and the ventrocaudal aspect of the nucleus of the lateral recess. The most conspicuous secretoneurin immunoreactivity was found in the magnocellular and parvocellular cells of the preoptic nucleus that project to the pituitary. Double-labeling studies indicated coexpression with isotocin, the fish homolog of mammalian oxytocin. Clear colabeling for secretoneurin and isotocin in fibers terminating in the neurointermediate lobe suggests that secretoneurin maybe coreleased with isotocin. Previous work indicates that secretoneurin stimulates the release of luteinizing hormone from the goldfish anterior pituitary. Our findings further support a reproductive role for secretoneurin and related peptides, given the importance of oxytocin family peptides in reproductive behavior in vertebrates.

Relationship between α-melanocyte-stimulating hormone- and neuropeptide Y-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) injection of α-melanocyte-stimulating hormone (α-MSH) inhibits, whereas ICV injection of neuropeptide Y (NPY) stimulates food intake in the goldfish. However, there is little information about the functional relationship between α-MSH-induced anorexigenic and NPY-induced orexigenic actions in the goldfish. In this study we examined the relationship between α-MSH- and NPY-containing neurons in the goldfish hypothalamus to investigate whether these α-MSH- and NPY-containing neurons have direct mutual inputs. α-MSH- and NPY-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. In particular, α-MSH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). NPY-containing nerve fibers or endings also lay in close apposition to α-MSH-containing neurons specifically in the interior part of the nucleus lateralis tuberis (NLTi). We also investigated the effect of ICV injection of melanocortin 4 receptor agonist (melanotan II) at 100 pmol/g body weight (BW), which is enough to suppress food intake, or NPY at 10 pmol/g BW, which is enough to enhance food intake, on expression levels of mRNA for NPY or proopiomelanocortin (POMC) in the hypothalamus. ICV injection of melanotan II and NPY induced a significant decrease in the expression levels for NPY and POMC mRNA, respectively. These observations suggest that α-MSH- and NPY-containing neurons share direct mutual inputs in the NPPv and the NLTi of the hypothalamus, and that α-MSH and NPY functionally interact or exhibit mutual inhibition to regulate feeding behavior in the goldfish.

Three Types of Gonadotrophin‐Releasing Hormone Neurones and Steroid‐Sensitive Sexually Dimorphic Kisspeptin Neurones in Teleosts

In general, the gonadotrophin-releasing hormone (GnRH) neuronal systems of vertebrates consist of one group of hypothalamic neuroendocrine and one or two group(s) of extrahypothalamic neuromodulatory GnRH neurones. By taking advantage of the brains of dwarf gourami and GnRH-green fluorescent protein transgenic medaka, the spontaneous electrical activities of all three different types of GnRH neurones have now been characterised. The hypophysiotrophic preoptic area-gnrh1 neurones show irregular and episodic spontaneous electrical activities, whereas extrahypothalamic midbrain gnrh2 and terminal nerve-gnrh3 neurones show regular pacemaker potentials. It is suggested that these spontaneous electrical activities are related to their different functions as neuroendocrine hormones (gnrh1 neurones) and neuromodulators (gnrh2 and gnrh3 neurones). On the other hand, recent evidence strongly suggests that the GnRH neurones are regulated by another class of peptidergic neurones, the kisspeptin neurones. The gene encoding kisspeptin (kiss1 gene) has been cloned, and the anatomical distribution of kiss1 mRNA expressing neurones (kiss1 neurones) has recently been studied in brains of several fish species. In medaka, two kiss1 neuronal populations in hypothalamic areas, called the nucleus ventral tuberis (NVT) and nucleus posterioris periventricularis (NPPv), have been identified. The NVT kiss1 neurones are sexually dimorphic in number (male >> female) under breeding conditions and are sensitive to ovarian oestrogens, whereas the NPPv kiss1 neurones are neither sexually dimorphic, nor sensitive to steroids. The steroid-sensitive changes in kiss1 mRNA expression in the NVT occur physiologically, closely linked to the reproductive state. As in the mammalian counterpart, the medaka kiss1 neuronal system is suggested to be involved in the central regulation of reproductive functions. Interestingly, kiss2, another gene paralogous to kiss1, has been cloned in some fish species. The possible regulation of reproduction and other unknown functions by kiss1 and kiss2 neurones may be the focus of future studies.

Neuronal relationship between orexin-A- and neuropeptide Y-induced orexigenic actions in goldfish

Orexin-induced orexigenic action is mediated by neuropeptide Y (NPY) in goldfish and rodents. A previous study indicated that NPY-induced orexigenic action may also be mediated by orexin-A in goldfish. However, there is little information about the mutual actions of orexin-A and NPY in the goldfish. Therefore, using their specific receptor antagonists, we examined whether the orexigenic actions of orexin-A and NPY mutually interact in the goldfish. The stimulatory effect of intracerebroventricular injection of NPY at 1 pmol/g body weight (BW) on food intake was abolished by treatment with the orexin receptor-1 antagonist, SB334867, at 10 pmol/g BW whereas the NPY Y1-receptor antagonist, BIBP3226, at 100 pmol/g BW attenuated orexin-A (at 2.8 pmol/g BW)-stimulated feeding. This led us, using a double-immunostaining method and confocal laser scanning microscopy, to investigate whether orexin-A- and NPY-containing neurons in the goldfish brain have direct mutual inputs. Orexin-A- and NPY-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. Orexin-A- and NPY-containing neurons were located in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv), in close proximity to each other: NPY-containing nerve fibers or endings lay in close apposition to orexin-A-containing neurons in the NPPv, and orexin-A-containing nerve fibers or endings also lay in close apposition to NPY-containing neurons in the same region. These results indicate that, in goldfish, orexin-A- and NPY-induced orexigenic actions are mediated by mutual signaling pathways.

Relationship between melanin-concentrating hormone- and neuropeptide Y-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike the orexigenic action in rodents, via the melanocortin system with suppression of neuropeptide Y (NPY) mRNA expression. We therefore investigated the neuronal relationship between MCH- and NPY-containing neurons in the goldfish brain, using a double-immunofluorescence method and confocal laser scanning microscopy. MCH- and NPY-like immunoreactivities were distributed throughout the brain. In particular, MCH-containing nerve fibers or endings lay in close apposition to NPY-containing neurons in a specific region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These observations suggest that MCH-containing neurons provide direct input to NPY-containing neurons in the NPPv of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide, inhibiting the orexigenic activity of NPY.

Interaction of orexin/hypocretin-like immunoreactive neurons with melanin-concentrating hormone and α-melanocyte-stimulating hormone neurons in brain of a pleuronectiform fish, barfin flounder

Immunohistochemical localization of orexin/hypocretin in the brain of a pleuronectiform fish, the barfin flounder Verasper moseri was examined as the first step in unraveling the possible function of the hormone in the brain. Orexin-A-like immunoreactive (ir) cell bodies were found to be located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-A-like-ir fibers were detected not only in the hypothalamus but also extensively throughout the brain. The orexin-A-like-ir cell bodies did not project their fibers to the pituitary gland. Since melaninconcentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are suggested to regulate food intake in addition to orexin/hypocretin in the teleost fish, it was examined whether neural connections exist between orexin neurons and the MCH and α-MSH neurons in the barfin flounder brain by using double-staining immunohistochemistry. Some orexin-A-like-ir fibers were in close contact with the MCH-ir and α-MSH-ir cell bodies in the hypothalamus. Moreover, a few MCH-ir and α-MSH-ir fibers were in close contact with the orexin-A-like-ir cell bodies in the hypothalamus. These results suggest that reciprocal connections exist between the orexin and MCH neurons and between the orexin and α-MSH neurons in the brain of the barfin flounder.

Corticotropin-releasing hormone mediates α-melanocyte-stimulating hormone-induced anorexigenic action in goldfish

α-Melanocyte-stimulating hormone (α-MSH) and corticotropin-releasing hormone (CRH) both suppress food intake, and the α-MSH- or CRH-signaling pathway has possible potency to mediate anorexigenic actions induced by most other neuropeptides in goldfish. Therefore, using specific receptor antagonists, we examined whether the anorexigenic actions of α-MSH and CRH mutually interact. The inhibitory effect of ICV injection of the α-MSH agonist, melanotan II (MT II), on food intake was abolished by treatment with a CRH 1/2 receptor antagonist, α-helical CRH (9–41), whereas the anorexigenic action of ICV-injected CRH was not affected by treatment with a melanocortin 4 receptor antagonist, HS024. This led us to investigate whether α-MSH-containing neurons in the goldfish brain have direct inputs to CRH-containing neurons, using confocal laser scanning microscopy. α-MSH- and CRH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. α-MSH-containing nerve fibers or endings lay in close apposition to CRH-containing neurons in a region of the hypothalamus, the nucleus posterioris periventricularis (NPPv). These results indicate that, in goldfish, α-MSH-induced anorexigenic action is mediated by the CRH-signaling pathway, and that CRH plays a crucial role in the regulation of feeding behavior as an integrated anorexigenic neuropeptide in this species.

Identification of KiSS-1 Product Kisspeptin and Steroid-Sensitive Sexually Dimorphic Kisspeptin Neurons in Medaka (Oryzias latipes)

Recently, a novel physiologically active peptide, kisspeptin (metastin), has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons in several mammalian species. Despite its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report on the kisspeptin or kisspeptin neuronal systems in nonmammalian vertebrates. We used medaka for the initial identification of the KiSS-1 gene and the anatomical distribution of KiSS-1 mRNA expressing neurons (KiSS-1 neurons) in the brain of nonmammalian species. In situ hybridization for the medaka KiSS-1 gene cloned here proved that two kisspeptin neuronal populations are localized in the hypothalamic nuclei, the nucleus posterioris periventricularis and the nucleus ventral tuberis (NVT). Furthermore, NVT KiSS-1 neurons were sexually dimorphic in number (male neurons >> female neurons) under the breeding conditions. We also found that the number of KiSS-1 neurons in the NVT but not that in the nucleus posterioris periventricularis was positively regulated by ovarian estrogens. The fact that there were clear differences in the number of NVT KiSS-1 neurons between the fish under the breeding and nonbreeding conditions strongly suggests that the steroid-sensitive changes in the KiSS-1 mRNA expression in the NVT occur physiologically, according to the changes in the reproductive state. From the present results, we conclude that the medaka KiSS-1 neuronal system is involved in the central regulation of reproductive functions, and, given many experimental advantages, the medaka brain may serve as a good model system to study its physiology.

Immunohistochemical localization of orexin/hypocretin-like immunoreactive peptides and melanin-concentrating hormone in the brain and pituitary of medaka

Orexin/hypocretin is a neuropeptide that is involved in the regulation of feeding behavior and the sleep-wakefulness cycle in mammals. Melanin-concentrating hormone (MCH) is believed to be another candidate involved in food intake in teleost fish as well. Thus, it is interesting to examine whether neural connections exist between the neurons producing these two hormones. We first examined the localization of orexin-like immunoreactivity (orexin-LI) in the brain of the medaka Oryzias latipes by using immunohistochemistry. We further examined the interaction between the orexin and MCH neurons in the medaka brain by performing double-staining immunohistochemistry. Orexin-LI cell bodies were located in the nucleus posterioris periventricularis (NPPv) of the hypothalamus, and orexin-LI fibers were detected not only in the hypothalamus but also extensively throughout the brain. Some orexin-LI fibers were in close contact with the MCH-immunoreactive (ir) cell bodies in the hypothalamus, as revealed by double-staining immunohistochemistry. Moreover, a few MCH-ir fibers were in close contact with the orexin-LI cell bodies. These results suggest that in the medaka brain, orexin performs various functions, including neuromodulation, and that neural connections exist between the orexin and MCH neurons.

Novel fish hypothalamic neuropeptides stimulate the release of gonadotrophins and growth hormone from the pituitary of sockeye salmon

We recently identified a cDNA encoding three novel fish hypothalamic neuropeptides, having LPXRF-NH(2) from the goldfish brain. In this study, to clarify the physiological functions of these three LPXRFamide peptides (gfLPXRFa-1, -2, and -3), we analysed the localisation and hypophysiotrophic activity of these peptides using sockeye salmon, Oncorhynchus nerka, in which immunoassay systems for several anterior pituitary hormones have been developed. gfLPXRFa-immunoreactive cell bodies were detected in the nucleus posterioris periventricularis of the hypothalamus and immunoreactive fibres were distributed in various brain regions and the pituitary. We also detected gfLPXRFa-immunoreactivity in the pituitary by competitive enzyme-linked immunosorbent assay combined with reversed-phase HPLC. These three gfLPXRFamide peptides stimulated the release of FSH, LH and GH, but did not affect the release of prolactin (PRL) and somatolactin (SL) from cultured pituitary cells. These results suggest that novel fish hypothalamic LPXR-Famide peptides exist in the brain and pituitary of sockeye salmon and stimulate the release of gonadotrophins and GH from the pituitary.

Novel fish hypothalamic neuropeptide.

Recently, we identified novel avian and amphibian hypothalamic neuropeptides that inhibited gonadotropin release and stimulated growth hormone release. They were characterized by a similar structure including the C-terminal LPLRF-NH2 motif. To clarify that the expression of these novel hypothalamic neuropeptides is a conserved property in vertebrates, we characterized a cDNA encoding a similar novel peptide, having LPLRF-NH2 from the goldfish brain, by a combination of 3' and 5' rapid amplification of cDNA ends (RACE). The deduced peptide precursor consisted of 197 amino acid residues, encoding three putative peptide sequences that included -LPXRF (where X is L or Q) at their C-termini. Mass spectrometric analyses revealed that a tridecapeptide (SGTGLSATLPQRF-NH2) was derived from the precursor in the brain as an endogenous ligand. Southern blotting analysis of reverse-transcriptase-mediated PCR products demonstrated a specific expression of the goldfish peptide gene in the diencephalon. In situ hybridization revealed the cellular localization of goldfish peptide mRNA in the nucleus posterioris periventricularis in the hypothalamus. Immunoreactive cell bodies were also restricted to the the nucleus posterioris periventricularis and the nervus terminalis and immunoreactive fibers were distributed in several brain regions including the nucleus lateralis tuberis pars posterioris and pituitary. Thus, the goldfish hypothalamus expresses a novel neuropeptide containing the C-terminal -LPQRF-NH2 sequence, which may possess multiple regulatory functions and act, at least partly, on the pituitary to regulate pituitary hormone release.