Aquaporin regulates cell rounding through vacuole formation during endothelial-to-hematopoietic transition.

Endothelial-to-hematopoietic transition (EHT) is crucial for hematopoietic stem cell (HSC) generation. During EHT, the morphology of hemogenic endothelial cells (HECs) changes from flat and adherent to spherical hematopoietic cells, which detach from the dorsal aorta. HECs attain a rounded shape in a mitosis-independent manner before cell adhesion termination, suggesting an atypical cell-rounding mechanism. However, the direct mechanisms underlying this change in cell morphology during EHT remain unclear. Here, we show that large vacuoles were transiently formed in avian HECs, and that aquaporin 1 (AQP1) was localized in the vacuole and plasma membranes. Overexpression of AQP1 in non-HECs induced ectopic vacuole expansion, cell rounding and subsequent cell detachment from the endothelium into the bloodstream, mimicking EHT. Loss of redundant AQP functions by CRISPR/Cas9 gene editing in HECs impeded the morphological EHT. Our findings provide the first evidence to indicate that morphological segregation of hematopoietic cells from endothelial cells is regulated by water influx into vacuoles. These findings provide important insights for further exploration of the mechanisms underlying cell/tissue morphogenesis through water-adoptive cellular responses.

Nanobody-based RFP-dependent Cre recombinase for selective anterograde tracing in RFP-expressing transgenic animals.

Transgenic animals expressing fluorescent proteins are widely used to label specific cells and proteins. By using a split Cre recombinase fused with mCherry-binding nanobodies or designed ankyrin repeat proteins, we created Cre recombinase dependent on red fluorescent protein (RFP) (Cre-DOR). Functional binding units for monomeric RFPs are different from those for polymeric RFPs. We confirmed selective target RFP-dependent gene expression in the mouse cerebral cortex using stereotaxic injection of adeno-associated virus vectors. In estrogen receptor-beta (Esr2)-mRFP1 mice and gastrin-releasing peptide receptor (Grpr)-mRFP1 rats, we confirmed that Cre-DOR can be used for selective tracing of the neural projection from RFP-expressing specific neurons. Cellular localization of RFPs affects recombination efficiency of Cre-DOR, and light and chemical-induced nuclear translocation of an RFP-fused protein can modulate Cre-DOR efficiency. Our results provide a method for manipulating gene expression in specific cells expressing RFPs and expand the repertory of nanobody-based genetic tools.

Neuronal pentraxin 2 is required for facilitating excitatory synaptic inputs onto spinal neurons involved in pruriceptive transmission in a model of chronic itch.

An excitatory neuron subset in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for pruriceptive transmission. Here, we show that glutamatergic excitatory inputs onto GRPR+ neurons are facilitated in mouse models of chronic itch. In these models, neuronal pentraxin 2 (NPTX2), an activity-dependent immediate early gene product, is upregulated in the dorsal root ganglion (DRG) neurons. Electron microscopy reveals that NPTX2 is present at presynaptic terminals connected onto postsynaptic GRPR+ neurons. NPTX2-knockout prevents the facilitation of synaptic inputs to GRPR+ neurons, and repetitive scratching behavior. DRG-specific NPTX2 expression rescues the impaired behavioral phenotype in NPTX2-knockout mice. Moreover, ectopic expression of a dominant-negative form of NPTX2 in DRG neurons reduces chronic itch-like behavior in mice. Our findings indicate that the upregulation of NPTX2 expression in DRG neurons contributes to the facilitation of glutamatergic inputs onto GRPR+ neurons under chronic itch-like conditions, providing a potential therapeutic target.

Vasopressin-oxytocin-type signaling is ancient and has a conserved water homeostasis role in euryhaline marine planarians.

Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.

Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at -25 °C for Several Years.

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Variation of pro-vasopressin processing in parvocellular and magnocellular neurons in the paraventricular nucleus of the hypothalamus: Evidence from the vasopressin-related glycopeptide copeptin.

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

Vasopressin gene products are colocalised with corticotrophin-releasing factor within neurosecretory vesicles in the external zone of the median eminence of the Japanese macaque monkey (Macaca fuscata).

Arginine vasopressin (AVP), when released into portal capillaries with corticotrophin-releasing factor (CRF) from terminals of parvocellular neurones of the hypothalamic paraventricular nucleus (PVH), facilitates the secretion of adrenocorticotrophic hormone (ACTH) in stressed rodents. The AVP gene encodes a propeptide precursor containing AVP, AVP-associated neurophysin II (NPII), and a glycopeptide copeptin, although it is currently unclear whether copeptin is always cleaved from the neurophysin and whether the NPII and/or copeptin have any functional role in the pituitary. Furthermore, for primates, it is unknown whether CRF, AVP, NPII and copeptin are all colocalised in neurosecretory vesicles in the terminal region of the paraventricular CRF neurone axons. Therefore, we investigated, by fluorescence and immunogold immunocytochemistry, the cellular and subcellular relationships of these peptides in the CRF- and AVP-producing cells in unstressed Japanese macaque monkeys (Macaca fuscata). Reverse transcription-polymerase chain reaction analysis showed the expression of both CRF and AVP mRNAs in the monkey PVH. As expected, in the magnocellular neurones of the PVH and supraoptic nucleus, essentially no CRF immunoreactivity could be detected in NPII-immunoreactive (AVP-producing) neurones. Immunofluorescence showed that, in the parvocellular part of the PVH, NPII was detectable in a subpopulation (approximately 39%) of the numerous CRF-immunoreactive neuronal perikarya, whereas, in the outer median eminence, NPII was more prominent (approximately 52%) in the CRF varicosities. Triple immunoelectron microscopy in the median eminence demonstrated the presence of both NPII and copeptin immunoreactivity in dense-cored vesicles of CRF-containing axons. The results are consistent with an idea that the AVP propeptide is processed and NPII and copeptin are colocalised in hypothalamic-pituitary CRF axons in the median eminence of a primate. The CRF, AVP and copeptin are all co-packaged in neurosecretory vesicles in monkeys and are thus likely to be co-released into the portal capillary blood to amplify ACTH release from the primate anterior pituitary.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice.

Two types of nuclear estrogen receptors, ERα and ERß, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERß expression, detailed anatomical distribution and neurochemical characteristics of ERß expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERß-RFPtg, in which RFP was inserted downstream of ERß BAC promotor. We verified RFP signals as ERß by confirming: (1) high ERß mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERß mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERß-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERß, and those expressing exclusively either ERα or ERß. The majority of PVN and DRN cells expressed only ERß-RFP. Further, ERß-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERß-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERß-RFPtg mice can be a powerful tool for future studies on ERß function in the estrogenic regulation of social behaviors.

The phosphatidylcholine transfer protein StarD7 is important for myogenic differentiation in mouse myoblast C2C12 cells and human primary skeletal myoblasts.

StarD7 is a phosphatidylcholine (PC)-specific lipid transfer protein essential for the maintenance of mitochondrial PC composition, morphogenesis, and respiration. Here, we studied the role of StarD7 in skeletal myoblast differentiation using mouse myoblast C2C12 cells and human primary myoblasts. Immunofluorescence and immuno-electron microscopy revealed that StarD7 was distributed in the cytosol, inner mitochondria space, and outer leaflet of the outer mitochondrial membrane in C2C12 cells. Unlike human kidney embryonic cell line HEK293 cells, the mitochondrial proteinase PARL was not involved in the processing and maturation of StarD7 in C2C12 cells. StarD7 was constantly expressed during myogenic differentiation of C2C12 cells. The siRNA-mediated knockdown of StarD7 in C2C12 cells and human primary myoblasts significantly impaired myogenic differentiation and reduced the expression of myomaker, myomerger and PGC-1α. The reduction in mitochondrial PC levels and oxygen consumption rates, decreased expression of myomaker, myomerger and PGC-1α, as well as impaired myogenic differentiation, were completely restored when the protein was reintroduced into StarD7-knockout C2C12 cells. These results suggest that StarD7 is important for skeletal myogenesis in mammals.

VGF in the Medial Preoptic Nucleus Increases Sexual Activity Following Sexual Arousal Induction in Male Rats.

The central part of the medial preoptic nucleus (MPNc) is associated with sexual arousal induction in male rats. However, it is largely unclear how males are sexually aroused and achieve their first copulation. We previously reported that more MPNc neurons activate during the first copulation than the second copulation. In this study, to explore the molecules responsible for sexual arousal induction, we performed DNA microarray of the MPNc in sexually naive males and males after they copulated for their first and second times. We then performed quantitative PCR analyses to validate the results of the DNA microarray. Six genes were identified. Their expression increased following copulation and was higher in males after they copulated for the first time than after the second time. The genes encode transcription factors (Fos, Nfil3, and Nr4a3), a serine/threonine kinase (Sik1), an antioxidant protein (Srxn1), and a neuropeptide precursor VGF (Vgf), which may be the candidate genes responsible for sexual arousal induction. We examined the effects of Vgf knockdown in the MPNc on sexual partner preference and sexual behavior in sexually inexperienced and experienced males to determine the role of VGF in sexual arousal induction. A preference for estrous female rats was reinforced, and the latency of mount and intromission became short after sexually inexperienced males copulated for the first time. However, Vgf knockdown disrupted these phenomena. Vgf knockdown did not have any significant effect in sexually experienced males. VGF-derived neuropeptides presumably serve as an effector molecule to increase sexual activity following sexual arousal induction.

Sexual experience reduces neuronal activity in the central part of the medial preoptic nucleus in male rats during sexual behavior.

The medial preoptic area (MPN) plays an important role in the control of male sexual behavior. In rats, the central part of the MPN (MPNc) is sexually dimorphic and contains a sexually dimorphic nucleus composed of neurons expressing calbindin-D28 K (CALB-SDN). Although the functions of the MPNc are not well understood, surgical destruction of the MPNc adversely affects the performance of sexual behavior in sexually naive males, but not in sexually experienced males, supporting the notion that the MPNc changes functionally with sexual experience. In this study, we aimed to determine the effects of sexual experience on the neuronal activity of the MPNc and CALB-SDN. Sexual behavior in sexually inexperienced males that had no experience of ejaculation, and experienced males that had ejaculated once was observed. After they displayed sexual behavior, the brains were sampled, and immunohistochemical analysis of c-Fos, a neuronal activity marker, in the MPNc and CALB-SDN was performed. The numbers of c-Fos-immunopositive cells with or without calbindin-D28K-immunoreactivity increased significantly in the MPNc and CALB-SDN following ejaculation in both sexually inexperienced and experienced males, although the numbers did not change significantly with exposure to estrous female odors, the first mount, and the first intromission before and after the first ejaculation. We further found that the number of c-Fos-immunopositive and calbindin-D28K-immunonegative cells in the MPNc, but not in the CALB-SDN, was significantly smaller in sexually experienced males than in sexually inexperienced males. These results suggest that a population of MPNc neurons, which is located outside the CALB-SDN and do not express calbindin-D28 K, are activated during the first copulation and then silent after acquisition of sexual experience.

Gonadal Hormone-Dependent Sexual Differentiation of a Female-Biased Sexually Dimorphic Cell Group in the Principal Nucleus of the Bed Nucleus of the Stria Terminalis in Mice.

We recently reported a female-biased sexually dimorphic area in the mouse brain in the boundary region between the preoptic area and the bed nucleus of the stria terminalis (BNST). We reexamined this area and found that it is a ventral part of the principal nucleus of the BNST (BNSTp). The BNSTp is a male-biased sexually dimorphic nucleus, but the ventral part of the BNSTp (BNSTpv) exhibits female-biased sex differences in volume and neuron number. The volume and neuron number of the BNSTpv were increased in males by neonatal orchiectomy and decreased in females by treatment with testosterone, dihydrotestosterone, or estradiol within 5 days after birth. Sex differences in the volume and neuron number of the BNSTpv emerged before puberty. These sex differences became prominent in adulthood with increasing volume in females and loss of neurons in males during the pubertal/adolescent period. Prepubertal orchiectomy did not affect the BNSTpv, although prepubertal ovariectomy reduced the volume increase and induced loss of neurons in the female BNSTpv. In contrast, the volume and neuron number of male-biased sexually dimorphic nuclei that are composed of mainly calbindin neurons and are located in the preoptic area and BNST were decreased by prepubertal orchiectomy but not affected by prepubertal ovariectomy. Testicular testosterone during the postnatal period may defeminize the BNSTpv via binding directly to the androgen receptor and indirectly to the estrogen receptor after aromatization, although defeminization may proceed independently of testicular hormones in the pubertal/adolescent period. Ovarian hormones may act to feminize the BNSTpv during the pubertal/adolescent period.

Sexual behavior-associated c-Fos induction in the sagittalis nucleus of the hypothalamus in male rat.

The sagittalis nucleus of the hypothalamus (SGN) is a small nucleus located in the interstitial area between the arcuate and ventromedial nuclei of the hypothalamus in rats. The SGN exhibits male-biased sexual dimorphism and expresses estrogen receptor α and calbindin-D28K. This suggests a contribution of the SGN to sexually differentiated brain function, but its functional role is unknown. In this study, neuronal activation in the SGN during sexual behavior in male rats was examined by c-Fos immunohistochemistry. The number of c-Fos-immunoreactive (c-Fos-ir) cells was elevated with only exposure to chemosensory cues of estrous females and significantly increased after the first mount. The first intromission and ejaculation did not induce further increases in the number of c-Fos-ir cells in the SGN. These findings suggest that the SGN is involved in regulation of the early phase of male sexual behavior, including motivation.

Arsenic Exposure Induces Unscheduled Mitotic S Phase Entry Coupled with Cell Death in Mouse Cortical Astrocytes.

There is serious concern about arsenic in the natural environment, which exhibits neurotoxicity and increases the risk of neurodevelopmental disorders. Adverse effects of arsenic have been demonstrated in neurons, but it is not fully understood how arsenic affects other cell types in the brain. In the current study, we examined whether sodium arsenite (NaAsO2) affects the cell cycle, viability, and apoptosis of in vitro-cultured astrocytes isolated from the cerebral cortex of mice. Cultured astrocytes from transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) were subjected to live imaging analysis to assess the effects of NaAsO2 (0, 1, 2, and 4 µM) on the cell cycle and number of cells. Fucci was designed to express monomeric Kusabira Orange2 (mKO2) fused with the ubiquitylation domain of hCdt1, a marker of G1 phase, and monomeric Azami Green (mAG) fused with the ubiquitylation domain of hGem, a marker of S, G2, and M phases. NaAsO2 concentration-dependently decreased the peak levels of the mAG/mKO2 emission ratio when the ratio had reached a peak in astrocytes without NaAsO2 exposure, which was due to attenuating the increase in the mAG-expressing cell number. In contrast, the mAG/mKO2 emission ratio and number of mAG-expressing cells were concentration-dependently increased by NaAsO2 before their peak levels, indicating unscheduled S phase entry. We further examined the fate of cells forced to enter S phase by NaAsO2. We found that most of these cells died up to the end of live imaging. In addition, quantification of the copy number of the glial fibrillary acidic protein gene expressed specifically in astrocytes revealed a concentration-dependent decrease caused by NaAsO2. However, NaAsO2 did not increase the amount of nucleosomes generated from DNA fragmentation and failed to alter the gene expression of molecules relevant to unscheduled S phase entry-coupled apoptosis (p21, p53, E2F1, E2F4, and Gm36566). These findings suggest that NaAsO2 adversely affects the cell cycle and viability of astrocytes by inducing unscheduled S phase entry coupled with cell death that may be caused by mechanisms other than apoptosis.

A neural connection between the central part of the medial preoptic nucleus and the bed nucleus of the stria terminalis to regulate sexual behavior in male rats.

The medial preoptic nucleus (MPN) is a regulatory center for male sexual behavior. It consists of sexually dimorphic structures that are male biased, and these structures are found in the central part of the MPN (MPNc). The bed nucleus of the stria terminalis (BNST) also participates in male sexual behavior, and receives efferent neural projections from the MPNc. In this study, we examined if MPNc neurons projecting to the BNST are activated in male rats displaying sexual behavior. Fluoro-Gold (FG; a retrograde neural tracer) was injected into the BNST of male rats, which were separated into two groups: (1) those in contact with estrous female rats and displayed sexual behavior followed by ejaculation and (2) those without contact with estrous female rats. In both groups, protein expression of c-Fos (a neuronal activity marker) and calbindin (a location marker of the MPNc) were detected by fluorescent immunohistochemistry. The number of c-Fos-immunoreactive cells with or without FG labeling in the MPNc was also measured. The number of c-Fos-immunoreactive cells significantly increased following ejaculation. Approximately 10% of FG-labeled cells in ejaculation male rats were immunoreactive for c-Fos, and this percentage value was significantly higher in this group compared with control male rats. Overall, these results suggest that efferent projections from the MPNc to the BNST function to control sexual behavior in male rats.

Characterization of an avian histidine decarboxylase and localization of histaminergic neurons in the chicken brain.

In mammals, it is established that histamine is a neurotransmitter and/or neuromodulator in the central nervous system. It is produced by the enzyme histidine decarboxylase (HDC) in the tuberomammillary nucleus of the posterior hypothalamus. However, HDC as well as histaminergic neurons have not yet been characterized in the avian brain. We have cloned the cDNA for HDC from the chicken hypothalamus and demonstrated that the chicken HDC sequence is highly homologous to the mammalian counterpart, and that the expressed protein shows high enzymatic activity. The expression of HDC mRNA at various sites in the brain was investigated using quantitative RT-PCR. The results showed that the HDC mRNA was highly expressed in the hypothalamic infundibulum. In situ hybridization analyses revealed that the cells containing HDC mRNA were localized in the medial mammillary nucleus of the hypothalamic infundibulum. Intracerebroventricular injection of histamine in chicks resulted in inhibition of feeding behavior. This is the first report of the characterization of histaminergic neurons in the avian brain, and our findings indicate that neuronal histamine exerts anorexigenic effects in chicks.

Identification of neurotensin and LANT-6 and localization of mRNA encoding their precursor in the chicken brain.

Neurotensin (NT) and neurotensin-related peptide (Lys(8), Asn(9), NT(8-13): LANT-6) have previously been purified from chicken intestine. However, the presence of these peptides and the localization of their precursor mRNA in the brain were not well understood. In the present study, through a comprehensive analysis of bioactive substances, NT and LANT-6 were identified in the chicken brain using tandem mass spectrometry combined with a bioassay of the colon contraction. The effect of NT and LANT-6 on the colon contraction was assessed, and NT was found to be 10 times more potent than LANT-6. Furthermore, the sites of NT/LANT-6 precursor mRNA expression in the brain were investigated using quantitative RT-PCR. The result showed that the mRNA was expressed most in the telencephalon, followed by the diencephalon. In situ hybridization analysis revealed that cells containing NT/LANT-6 precursor mRNA were widely distributed throughout the brain except for the cerebellum. Additionally, these were highly concentrated in the frontal telencephalon, including the nidopallium, hyperpallium, and hippocampus. Collectively, these results indicate that NT and LANT-6 are produced in the chicken brain, and they may participate in multiple functions.

Identification of a cDNA encoding a novel small secretory protein, neurosecretory protein GL, in the chicken hypothalamic infundibulum.

To find novel neuropeptide and/or peptide hormone precursors in the avian brain, we performed a cDNA subtractive screen of the chicken hypothalamic infundibulum, which contains one of the feeding and neuroendocrine centers. After sequencing 596 clones, we identified a novel cDNA encoding a previously unknown protein. The deduced precursor protein consisted of 182 amino acid residues, including one putative small secretory protein of 80 amino acid residues. This small protein was flanked at the N-terminus by a signal peptide and at the C-terminus by a glycine amidation signal and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the small protein were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated specific expression of the NPGL precursor mRNA in the hypothalamic infundibulum. Furthermore, the mRNA levels in the hypothalamic infundibulum increased during post-hatching development. In situ hybridization analysis showed that the cells containing the NPGL precursor mRNA were localized in the medial mammillary nucleus and infundibular nucleus within the hypothalamic infundibulum of 8- and 15-day-old chicks. Subcutaneous infusion of NPGL in chicks increased body weight gain without affecting food intake. To our knowledge, this is the first report to describe the identification and localization of the NPGL precursor mRNA and the function of its translated product in animals. Our findings indicate that NPGL may participate in the growth process in chicks.

Changes in plasma angiotensin II, aldosterone, arginine vasotocin, corticosterone, and electrolyte concentrations during acclimation to dry condition and seawater in the crab-eating frog.

The crab-eating frog Fejervarya cancrivora inhabits mangrove swamps and marshes in Southeast Asia. In the present study, circulating angiotensin II (Ang II), aldosterone (Aldo), arginine vasotocin (AVT), and corticosterone (Cort) concentrations as well as various blood parameters were studied under osmotically stressful conditions. Following acclimation to hyperosmotic seawater and dry condition for 5days, body weight was significantly decreased. Under both conditions, plasma Na(+), Cl(-), and urea concentrations, hematocrit values (Ht; blood volume indicator), and osmolality were significantly increased. Dehydration associated with hypovolemic and hyperosmotic states of body fluids was induced during acclimation to hyperosmotic seawater and dry condition in the crab-eating frogs. Ang II, Aldo, AVT, and Cort were maintained within relatively narrow concentration ranges in the control frogs; however, in frogs under dry and hyperosmotic seawater conditions, large variations were observed among individuals in each group. Mean plasma Ang II and Aldo concentrations significantly increased in hyperosmotic seawater-acclimated and desiccated frogs. Although mean plasma AVT concentrations in dehydrated frogs of both the groups were approximately 2.0-3.5 times higher than those in the control frogs, the differences were not significant because of the variation. There was a significant correlation between plasma osmolality and AVT as well as Ang II but not Aldo. A significant correlation was also observed between Ht and AVT as well as Ang II. Plasma Ang II was significantly correlated with plasma Aldo. These results indicate that the crab-eating frogs may exhibit similar physiological responses to both seawater-acclimated and dry conditions. It appears that under dehydrated conditions, osmoregulatory mechanisms participate in stabilization of the situation. The renin-angiotensin system may have pivotal roles in body fluid regulation under volemic and osmotic stress in the Fejervarya species with unique osmoregulation.

The epithelial sodium channel in the Australian lungfish, Neoceratodus forsteri (Osteichthyes: Dipnoi).

Epithelial sodium channel (ENaC) is a Na(+)-selective, aldosterone-stimulated ion channel involved in sodium transport homeostasis. ENaC is rate-limiting for Na(+) absorption in the epithelia of osmoregulatory organs of tetrapods. Although the ENaC/degenerin gene family is proposed to be present in metazoans, no orthologues or paralogues for ENaC have been found in the genome databases of teleosts. We studied full-length cDNA cloning and tissue distributions of ENaCα, ß and γ subunits in the Australian lungfish, Neoceratodus forsteri, which is the closest living relative of tetrapods. Neoceratodus ENaC (nENaC) comprised three subunits: nENaCα, ß and γ proteins. The nENaCα, ß and γ subunits are closely related to amphibian ENaCα, ß and γ subunits, respectively. Three ENaC subunit mRNAs were highly expressed in the gills, kidney and rectum. Amiloride-sensitive sodium current was recorded from Xenopus oocytes injected with the nENaCαßγ subunit complementary RNAs under a two-electrode voltage clamp. nENaCα immunoreactivity was observed in the apical cell membrane of the gills, kidney and rectum. Thus, nENaC may play a role in regulating sodium transport of the lungfish, which has a renin-angiotensin-aldosterone system. This is interesting because there may have been an ENaC sodium absorption system controlled by aldosterone before the conquest of land by vertebrates.