Abstract
We examined the effects of i.c.v. administration of neuro-peptide W-30 (NPW30) on plasma arginine vasopressin (AVP) and plasma oxytocin (OXT) using RIA. The induction of c-fos mRNA, AVP heteronuclear (hn)RNA, and c-Fos protein (Fos) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of rats were also investigated using in situ hybridization histochemistry for c-fos mRNA and AVP hnRNA, and immunohistochemistry for Fos. Both plasma AVP and OXT were significantly increased at 5 and 15 min after i.c.v. administration of NPW30 (2.8 nmol/rat). In situ hybridization histochemistry revealed that the induction of c-fos mRNA and AVP hnRNA in the SON and PVN were significantly increased 15, 30, and 60 min after i.c.v. administration of NPW30 (1.4 nmol/rat). Dual immunostaining for Fos/AVP and Fos/OXT revealed that both AVP-like immunoreactive (LI) cells and OXT-LI cells exhibited nuclear Fos-LI in the SON and PVN, 90 min after i.c.v. administration of NPW30 (2.8 nmol/rat). These results suggest that central NPW30 may be involved in the regulation of secretion of AVP and OXT in the magnocellular neurosecretory cells in the SON and PVN.
Introduction
Neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30), 23- and 30-amino acid neuropeptide respectively, were originally isolated from porcine hypothalamus (Shimomura et al. 2002). The amino acid sequence of NPW23 is identical to that of the 23 residues of the N-terminal in NPW30 (Shimomura et al. 2002). These neuropeptides were discovered as endogenous ligands of G protein-coupled orphan receptors, GPR7 and GPR8, which were originally identified by the cloning of opioid-somatostatin-like receptor genes from human genomic DNA (O’Dowd et al. 1995). NPWs and GPR7 are widely distributed in the central nervous system (CNS) Lee et al. 1999, Dun et al. 2003).
The i.c.v. administration of NPW23 and NPW30 change secretion of anterior pituitary hormones, such as prolactin and growth hormone, and behavior, such as feeding and drinking (Shimomura et al. 2002, Baker et al. 2003, Mondal et al. 2003, Ishii et al. 2003, Levine et al. 2005, Taylor et al. 2005). NPW23 and NPW30 bind to and activate their receptors at similar effective doses (Mondal et al. 2003).
NPW23-like immunoreactive (LI) cells and GPR7 are detected in the parvocellular divisions of the paraventricular nucleus (PVN), which contain neurosecretory cells that produce corticotropin-releasing hormone (CRH) in rats (Lee et al. 1999, Dun et al. 2003). In rats, i.c.v. administration of NPW23 stimulated the secretion of corticosterone and activated the putative CRH-producing neurons in the parvocellular division of the PVN (Baker et al. 2003, Taylor et al. 2005). Endogenous NPW may have physiological roles in the neuroendocrine response to stress (Taylor et al. 2005).
The overall distribution of Fos-LI cells after i.c.v. administration of NPW23 was consistent with the distribution of GPR7 receptors (Levine et al. 2005). In detail, Fos-LI cells after i.c.v. administration of NPW23, NPW23-LI cells, and GPR7 are detected in the supraoptic nucleus (SON), which contains magnocellular neurosecretory cells (MNCs) that produce arginine vasopressin (AVP) and oxytocin (OXT) in rats (Lee et al. 1999, Dun et al. 2003, Levine et al. 2005). However, there are few studies that examine the effects of NPWs on the secretion of AVP and OXT in plasma and MNCs in the SON (Taylor et al. 2005).
In the present study, we first examined the effects of i.c.v. administration of NPW30 on plasma concentrations of AVP and OXT in rats using RIA. Secondly, we examined whether i.c.v. administration of NPW30 activated MNCs in the SON and PVN using in situ hybridization histochemistry for c-fos mRNA. Finally, whether i.c.v. administration of NPW30 selectively activated AVP- and OXT-producing MNCs in the SON and PVN was determined using dual immunostaining for Fos/AVP and Fos/OXT. The expression of the c-fos gene has been widely used to detect the neuronal activity in the CNS, in particular, the MNCs in the SON and PVN (Sagar et al. 1988, Ceccatelli et al. 1989, Sharp et al. 1991, Handa et al. 1993, Larsen & Mikkelsen 1995, Kawasaki et al. 2005). The MNCs in the PVN as well as in the SON are known to synthesize AVP and OXT, which are released into general circulation through terminal axons located in the posterior pituitary. In addition, intronic in situ hybridization histo-chemistry for AVP heteronuclear (hn)RNA was performed to detect the effects of i.c.v. administration of NPW30 on the transcription of the AVP gene in the SON and PVN because it has been used to detect rapid changes in AVP gene transcription (Herman et al. 1991, Priou et al. 1993, Arima et al. 1999, Kawasaki et al. 2005).
Materials and Methods
Animals
Adult male Wistar rats, weighing 295±2.0 g (mean±S.E.M.), were used in all experiments. They were housed individually in standard plastic cages in an air-conditioned room (23–25 ° C) under a 12 h light (0700–1900 h):12 h darkness (1900–0700 h) cycle. Food and water were available ad libitum.
Surgical procedures
For i.c.v. administration of NPW30 or saline, the animals were anesthetized (sodium pentobarbital, 50 mg/kg body weight) by i.p. injection and then placed in a stereotaxic frame in a prone position. A stainless steel guide cannula (550 μm outer diameter, 10 mm length) was implanted stereotaxically, following coordinates given by Paxinos & Watson (1986). These coordinates were 0.8 mm posterior to the bregma, 1.4 mm lateral to midline, and 2.0 mm below the surface of the right cortex, so that the tip of the cannula was 1.0 mm above the right cerebral ventricle. Two stainless steel anchoring screws were fixed to the skull, and the cannula was secured in place by acrylic dental cement. The rodents were then returned to their cages and allowed to recover for at least 7 days. They were then handled every day and housed in cages before the start of the experiments.
Central administration of NPW30 or vehicle
For i.c.v. administration of NPW30, a stainless steel injector (300 μm outer diameter) was introduced through the cannula to a depth of 1.0 mm beyond the end of the guide. The total volume of solution injected into the lateral ventricle was 10 μl. This volume was also used in previous studies (Serino et al. 1999, Shen & Krukoff 2001, Ozaki et al. 2002, Hashimoto et al. 2005). Rat NPW30 was purchased from the Peptide Institute (Osaka, Japan). The quality of rat NPW30 has been confirmed by Peptide Institute (supplementary data in the online version of Journal of Endocrinology at http:// joe.endocrinology-journals.org/content/vol190/issue2/). NPW30 was dissolved in sterile pyrogen-free 0.9% saline solution (Otsuka Pharmaceutical Co., Ltd, Tokyo, Japan).
Experimental procedures
In the first experiment, i.c.v. administration of NPW30 (1.4 and 2.8 nmol/rat) or 0.9% saline (vehicle) was performed (n=6 in each group). The rodents were decapitated 5, 15, 30, and 60 min after i.c.v. administration. The trunk blood was collected in plastic tubes containing 0.1 ml of heparin (200 U/ml; Sigma) and then spun (3000 r.p.m.) at 4 ° C for 10 min before the plasma concentrations of AVP and OXT were measured using a RIA.
In the second experiment, NPW30 (0.3, 1.4, and 2.8 nmol/rat) or vehicle was also administered i.c.v. (n=6 in each group). Thirty minutes after i.c.v. injection, the rodents were decapitated. Brains were removed and placed on powdered dry ice for in situ hybridization histochemistry for c-fos mRNA.
In the third experiment, i.c.v. administration of NPW30 (1.4 nmol/rat) or vehicle was performed (n=6 in each group). The rodents were decapitated 5, 15, 30, 60, or 180 min after i.c.v. administration. The time-course of this experiment was determined by our previous studies (Ozaki et al. 2002, Hashimoto et al. 2005, Kawasaki et al. 2005). Brains were removed and placed on powdered dry ice for in situ hybridization histochemistry for c-fos mRNA and AVP hnRNA.
In the final experiment, NPW30 (2.8 nmol/rat) or vehicle was administered i.c.v. (n=5 in each group). Ninety minutes after i.c.v. administration of the solution, the rodents were anesthetized deeply with an i.p. injection of sodium pentobarbital (75 mg/kg body weight) following perfusion and then fixed brains were used for immunohistochemistry for Fos, AVP, and OXT.
All experimental procedures in this study were performed in accordance with guidelines on the use and care of laboratory animals as set out by the Physiological Society of Japan and under the control of the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan.
RIA for AVP and OXT
Plasma concentrations of AVP and OXTwere determined by RIA with specific anti-AVP (Sakurai et al. 1985) and anti-OXT (Higuchi et al. 1985) antiserum as described previously (Onaka & Yagi 1990). The inter- and intraassay coefficients of variation were 14 and 7% for AVP, and 10 and 6% for OXT respectively. The minimum detection limits were 0.48 and 7.8 pg/ml for AVP and OXT respectively.
In situ hybridization histochemistry for c-fos mRNA and AVP hnRNA
All rodents were decapitated between 1400 and 1700 h, the brains were removed rapidly, frozen on dry ice, and stored at −80 ° C. Frozen sections were cut at 12 μm-thickness in a cryostat at −20 ° C and mounted onto gelatin/chrome alum-coated slides. The SON and PVN were localized by referring to the standard atlas of the rat brain by Paxinos & Watson (1986). The sections including the SON were chosen from plate 23 in the atlas. The sections including the PVN were chosen from plate 25 in the atlas. The localization of the sections obtained from each rat was checked for similar positions by microscopic observation. The SON and PVN consist of four sections, i.e., eight sites per rat, were used to measure the density of signals by autoradiography. The slides were warmed to room temperature and allowed to dry for 10 min and then fixed in 4% formaldehyde in phosphate buffered saline (PBS) for 5 min. They were then washed twice in PBS and incubated in 0.9% NaCl containing 0.25% acetic anhydride (v/v) and 0.1 M triethanolamine at room temperature for 10 min. The sections were then dehydrated using a series of 70% (1 min), 80% (1 min), 95% (2 min), and 100% (1 min) ethanol solutions consecutively and dilapidated in 100% chloroform for 5 min. The slides were then rehydrated partially first in 100% (1 min) and then 95% (1 min) ethanol and allowed to air dry briefly.
Hybridization was carried out overnight at 37 ° C in 45 μl hybridization buffer containing 50% formamide and 4× saline sodium citrate (SSC; 1× SSC=150 mM NaCl and 15 mM sodium citrate), which contained 500 μg/ml sheared salmon sperm DNA (Sigma), 250 μg/ml baker’s yeast total RNA (Boehringer Mannheim GmbH, Mannheim, Germany), 1× Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrlidone, and 0.02% BSA), and 10% dextransulfate (molecular mass 500 000 Da; Sigma) under a Nescofilm (Bando Chemical IMD, Ltd, Osaka, Japan) coverslip. The probes used were 3′-end 35S-oligodeoxynucleotides complementary to mRNA cording for c-fos (probe sequence=5′-CAG CGG GAG GAT GAC GCC TCG TAG TCC GCG TTG AAA CCC GAG AAC ATC-3′) and AVP hnRNA (probe sequence=5′-GCA CTG TCA GCA GCC CTG AAC GGA CCA CAG TGG TAC-3′). The specificity of each probe has been described previously (Nomura et al. 1999, Serino et al. 1999). Total counts of 1× 106 c.p.m./slide were used. After hybridization, sections were washed for 1 h at intervals of 15 min in 1× SSC at 55 ° C, and for a further 1 h at intervals of 30 min in 1× SSC at room temperature. All independent experimental sections were treated simultaneously to minimize the variable effects of hybridization and wash stringency. Hybridized sections containing the SON and PVN were apposed to autoradiography film (Hyperfilm; Amersham) for 10 days for each probe. The autoradiographic images were quantified using an MCID imaging analyzer (Imaging Research, Inc., St Catherines, ON, Canada). The images were captured by charge-coupled device camera (DAGE-MTI, Inc., Michigan City, IN, USA) with 40× magnification.
The locations of the magnocellular division of the SON and the magnocellular and parvocellular divisions of the PVN were chosen from the atlas. The areas of the SON and PVN were enclosed using captured images. The edges of the SON and PVN were determined using plates 23 and 25 of the atlas respectively (Paxinos & Watson 1986). The areas of the PVN contained both the magnocellular and parvocellular divisions. The mean optical density of autoradiographs was measured by comparing it with simultaneously exposed [14C] microscale samples (Amersham). The standard curve was fitted by the optical density of the [14C] microscale in the same film. Slides hybridized with the probes of the c-fos mRNA and AVP hnRNA were dipped into a nuclear emulsion (K-5, Ilford, Cheshire, UK) and exposed for a further 30 days. After development, the sections that stained by cresylecht violet were observed by microscopy.
Dual detection of Fos-LI and AVP-/OXT-LI
Deeply anesthetized animals were perfused transcardially with 0.1 M phosphate buffer (PB; pH 7.4) containing heparin (1000 U/l) followed by 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB. The brains were then removed and divided into three blocks that included the hypothalamus. The blocks were post-fixed with 4% paraformaldehyde and 0.2% picric acid in 0.1 M PB for 60 h at 4 ° C. The tissues were then cryoprotected in 20% sucrose in 0.1 M PB for 48 h at 4 ° C. Serial sections of 30 μm for dual staining for Fos and AVP/OXT were cut using a microtome. The sections were rinsed twice with 0.1 M PBS containing 0.3% Triton X-100 with 1% hydrogen peroxidase for 60 min. They were then rinsed twice with 0.1 M PBS containing 0.3% Triton X-100. The floating sections were incubated with a primary Fos antibody (sc-52, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), followed by a dilution ratio of 1:500 in 0.1 M PBS containing 0.3% Triton X-100 at 4 ° C for 3 days. After washing for 20 min in 0.1 M PBS solution containing 0.3% Triton X-100, the sections were further incubated for 120 min with a biotinylated secondary antibody solution (1:250), and finally with an avidin–biotin peroxidase complex (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA, USA) for 120 min. The peroxidase in the sections was visualized with 0.02% diaminobenzidine in a Tris buffer containing 0.05% hydrogen peroxidase for 5–7 min. During the dual staining for AVP or OXT, the sections were incubated sequentially in AVP antibody (INCSTAR Corp., Stillwater, MN, USA; diluted 1:10 000) or OXT antibody (CHEMICON International, Inc., Temecula, CA, USA; diluted 1:5000) for 2 days each at 4 ° C. The avidin–biotin peroxidase complex was made visible using nickel sulfate.
The sections were then mounted onto gelatin-coated slides, air dried, dehydrated in 100% ethanol, cleared with xylene, and coverslipped. The presence of a dark-brown label that appeared in round structures was judged as indicative of Fos-LI positive nuclei and a violet label that appeared in spindle-shaped structures was judged as indicative of AVP-/OXT-LI. Details of the immunohistochemistry have been published elsewhere (Ison et al. 1993, Onaka et al. 1995). To count the double-labeled cells, three serial sections including the SON per animal and two serial sections including the PVN per animal were chosen and counted under a light microscope.
Statistical analysis
A mean deviation from control (percentage)±S.E.M. was calculated from the results of the in situ hybridization histochemistry. Each group within an experiment was compared to the control group. The results of RIA and immunohistochemical staining were also expressed as the mean±S.E.M. The data were analyzed using a one-way fractional ANOVA followed by a Fisher’s-protected least significant difference type adjustment test for multiple comparisons. The statistical significance was set at P< 0.05.
Results
Effects of i.c.v. administration of NPW30 on plasma concentrations of AVP and OXT
The concentrations of plasma AVP and OXT were measured after i.c.v. administration of NPW30 (1.4 and 2.8 nmol/rat) or vehicle. In comparison to vehicle, the concentrations of both plasma AVP and OXT were significantly increased in a dose-related manner, 5 min after i.c.v. administration of NPW30 (1.4 and 2.8 nmol/rat), and were also significantly increased 15 min after i.c.v. administration of NPW30 (2.8 nmol/rat; Fig. 1; n=6 in each group).
Effects of i.c.v. administration of NPW30 on the expression of the c-fos gene in the SON and PVN
In situ hybridization histochemistry revealed that the expression of the c-fos gene in the SON and PVN increased 30 min after i.c.v. administration of NPW30, in all the doses (0.3, 1.4, and 2.8 nmol/rat; Fig. 2). Thirty minutes after administration of NPW30 (0.3 nmol/rat), the expression of the c-fos gene increased significantly in the SON (143±11% of control; n=6 in each group; P=0.03; Fig. 2A) and PVN (395±17% of control; n=6 in each group; P<0.01; Fig. 2B). Thirty minutes after i.c.v. administration of NPW30 (1.4 nmol/rat), the expression of the c-fos gene increased significantly in the SON (330±16% of control; n=6 in each group; P<0.05; Fig. 2A) and PVN (523±22% of control; n=6 in each group; P<0.01; Fig. 2B). Thirty minutes after i.c.v. administration of NPW30 (2.8 nmol/rat), the expression of the c-fos gene increased significantly in the SON (358±19% of control; n=6 in each group; P<0.05; Fig. 2A) and PVN (596±27% of control; n=6 in each group; P<0.01; Fig. 2B).
In situ hybridization histochemistry also revealed that the i.c.v. administration of NPW30 (1.4 nmol/rat) caused significant increases in the expression of the c-fos gene from 15 to 60 min in the SON and PVN (Fig. 3). The i.c.v. administration of NPW30 induced the expression of the c-fos gene to the greatest degree at 30 min in the SON (450±48% of control at 5 min; n=6 in each group; P<0.01; Fig. 3A) and PVN (666±68% of control at 5 min; n=6 in each group; P<0.01; Fig. 3B). The expression of the c-fos gene after i.c.v. administration of vehicle did not change significantly. Microscopic observation revealed that the c-fos gene-expressing cells were distributed throughout the SON (Fig. 4A and C) and in the parvocellular and magnocellular divisions of the PVN throughout the ependymal cells of the third ventricle (Fig. 4B and D). The sections stained by cresylecht violet also emphasized the distributions of the c-fos gene-expressing cells in the SON and PVN (Fig. 4E and F).
Dual detection of Fos-LI and OXT-/AVP-LI in the SON and PVN
Numerous Fos-LI cells in the SON after i.c.v. administration of NPW30 (2.8 nmol/rat) were concomitantly labeled with one of either antibody for AVP or OXT (Fig. 5). In the magnocellular part of the PVN, numerous Fos-LI cells were also concomitantly labeled with one of either antibody for AVP or OXT (Fig. 6). Taking the total AVP-LI-positive cells counted in each nucleus as 100%, the percentage of Fos-LI-positive cells was 59.3±5.4% in the SON and 71.9± 2.6% in the PVN after i.c.v. administration of NPW30 (2.8 nmol/rat, n=5). After i.c.v. administration of vehicle, the percentage of Fos-LI-positive cells of AVP-LI-positive cells was 6.77±3.7% in the SON and 19.3±5.2% in the PVN (n=5). Similarly, the percentage of Fos-LI-positive cells of OXT-LI was 85.4±3.3% in the SON and 90.1±1.5% in the PVN, after i.c.v. administration of NPW30 (2.8 nmol/rat, n=5). After i.c.v. administration of vehicle, the percentage of Fos-LI-positive cells of OXT-LI was 9.40±0.99% in the SON and 29.5±1.3% in the PVN (n=5). The distributions of the dual-stained cells are shown in Fig. 7 and 8. Although the Fos-LI cells were detected throughout the SON and PVN, the Fos-LI-positive cells of AVP-LI cells were found throughout the SON and mainly in the magnocellular divisions of the PVN. The Fos-LI-positive cells of OXT-LI cells were also found throughout the SON and in the anterior magnocellular and ventral medial parvocellular divisions of the PVN.
Effects of i.c.v. administration of NPW30 on AVP hnRNA in the SON and PVN
In situ hybridization histochemistry revealed that the i.c.v. administration of NPW30 (1.4 nmol/rat) caused significant increases in the expression level of AVP hnRNA from 15 to 60 min in the SON and PVN (Fig. 9). The i.c.v. administration of NPW30 induced the expression level of AVP hnRNA to the greatest degree at 30 min in the SON (167±7.4% of control at 5 min; n=6 in each group; P< 0.01; Fig. 9A) and PVN (205±11% of control at 5 min, n=6 in each group, P<0.01; Fig. 9B). The expressing cells of AVP hnRNA were observed throughout the SON (Fig. 10A and C) and in the magnocellular and parvocellular divisions of the PVN (Fig. 10B and D). The sections stained by cresylecht violet also emphasized the distributions of the expressing cells of AVP hnRNA in the SON and PVN (Fig. 10E and F).
Discussion
The present study demonstrated that centrally administered NPW30 caused significant increases in the plasma AVP and OXT levels in conscious rats. Centrally administered NPW30 also induced the c-fos gene expression throughout the SON and the magnocellular and parvocellular neurons of the PVN. Dual immunohistochemistry confirmed that most AVP- and OXT-LI cells expressed Fos-LI in the SON and PVN, after central administration of NPW30.
The plasma concentrations of AVP and OXT were increased significantly 5 and 15 min after i.c.v. administration of NPW30 (2.8 nmol/rat) and returned to normal levels since then (Fig. 1). Ozaki et al.(2002) also demonstrated that centrally administered neuromedin U (NMU), a peptide that suppresses food intake, caused significant increases in the plasma concentration of OXT from 15 to 60 min after i.c.v. administration of NMU in conscious rats. The effect of i.c.v. administration NMU on plasma OXT levels appeared later than those of NPW30. Later elevations of plasma OXT levels after i.c.v. administration of NMU may be mediated, at least in part, by secondary effects, such as changes in behavior (Howard et al. 2000), hyperthermia (Nakazato et al. 2000), and increased arterial blood pressure (Minamino et al. 1985). Relatively rapid changes in plasma AVP and OXT levels after i.c.v. administration of NPW30 indicate that centrally administered NPW30 may activate AVP- and OXT-secreting neurons directly or the neural pathways to these neurons. In addition, it may be expected that degradation and/or clearance of NPW30 in the cerebrospinal fluid may be shorter and/or faster than that of NMU, because increase in plasma AVP and OXT levels was rapid and transient (5–15 min) after i.c.v. administration of NPW30 compared with those results obtained from i.c.v. administration of NMU. The secondary effects, such as possible changes of arterial blood pressure and/ or plasma osmolality, may affect the plasma AVP and OXT levels after i.c.v. administration of NPW30, however, those possibilities can be excluded, because Taylor et al.(2005) showed centrally administered NPW23 did not alter arterial blood pressure or heart rate, and we have demonstrated that the plasma osmolality at any time (5, 15, 30, and 60 min) in the time-course study and any dose (1.4 and 2.8 nmol) in the dose–response study used here did not change significantly after i.c.v. administration of vehicle or NPW (data not shown).
Taylor et al.(2005) have demonstrated that plasma concentrations of AVP and OXT were not increased significantly, 15 min after i.c.v. administration of NPW23 (1 and 3 nmol/rat, 2 μl volume of total solutions) in conscious rats. They have also demonstrated that the release of AVP and OXT were not significantly increased 5 and 15 min after i.c.v. administration of neuropeptide B (NPB), which was also discovered as endogenous ligand of GPR7 (Samson et al. 2004). It is possible to explain why the effects of NPW23 and NPW30 in the present study, and NPB on the concentrations of AVP and OXT in plasma were different. One possibility is the biochemical difference among NPW23, NPW30, and NPB. Although NPWs and NPB are expressed from two different genes, these peptides share approximately 60% sequence homology (Fujii et al. 2002). NPW23 and NPW30 bind to and activate their receptors at similar effective dose, and also, NPWs and NPB bound with similar affinity to their receptors (Mondal et al. 2003, Tanaka et al. 2003). Biochemical differences alone cannot explain this discrepancy; the reason for the early elevations of plasma AVP and OXT after i.c.v. administration of NPW30 remains unclear. Another possibility is the difference in the volume of centrally administered solutions. Although they used a small volume (2 μl) for i.c.v. administration, we used a relatively large volume (10 μl). We cannot exclude the possibility that a relatively large volume of centrally administered solution may reach and activate wide brain sites, such as the brainstem, which is known to terminate their axons to the MNCs in the SON and PVN.
Although i.c.v. administration of NPW30 caused an increase in plasma AVP in conscious rats, its physiological meaning is not clear. A previous study demonstrated that central administration of NPW23 stimulated drinking in rats (Baker et al. 2003). It is postulated that NPW in the CNS may be involved in the regulation of AVP secretion, drinking behavior, and body fluid balance. OXT is a peptide, which has various kinds of physiological functions; however, we could not suggest in this study the physiological relevance to the transient elevation of plasma OXT. Further physiological and behavioral studies need be performed to elucidate the relationship between NPW-induced elevation of OXT and physiological/behavioral changes.
Dual immunostaining showed that Fos-LI was seen in both AVP-LI and OXT-LI cells throughout the SON and in the magnocellular and parvocellular divisions of the PVN. A previous study demonstrated that GPR7, which is the receptor bound to and activated by NPW30, was observed in the various brain regions, including the SON and parvocel-lular division of the PVN (Lee et al. 1999). Our results showed that the distribution of the Fos-LI cells appears to overlap with that of GPR7 mRNA in the SON and the parvocellular division of the PVN. An electrophysiological study demonstrated that NPW acted directly on the post-synaptic membrane of the parvocellular neurosecretory cells in the PVN, using the whole-cell patch clamp technique by perfusion with tetrodotoxin (Taylor et al. 2005). Our results showed that Fos-LI cells were detected not only in the parvocellular division, but also in the magnocellular division of the PVN. As Fos is directly and/or indirectly induced by an activation of neuronal activity, the parvocellular neuro-secretory cells in the PVN are activated directly, and the MNCs in the PVN may be activated indirectly after i.c.v. administration of NPW30. The primary site activated by i.c.v. administration of NPW30 in the CNS is not clear, except for the parvocellular neurosecretory cells in the PVN. The large number of Fos/AVP cells and Fos/OXT MNCs were observed in the SON and PVN after i.c.v. administration of NPW30. These results are consistent with the results that elevations in plasma concentrations of both AVP and OXT were observed after i.c.v. administration of NPW30.
In the present study, we demonstrated that the expression levels of the c-fos mRNA and Fos-LI in the SON and PVN were increased after i.c.v. administration of NPW30. The marked increase in the c-fos mRNA in the SON and PVN was observed 30 min after i.c.v. administration of NPW30. The increase in the expression level of c-fos mRNA should reflect the neuronal activation either directly and/or indirectly after i.c.v. administration of NPW30. The neuronal activity in the SON and PVN may be modulated by the NPW30 released in an autocrine and/or paracrine manner, because NPW-LI cells were detected in the SON and PVN (Dun et al. 2003). Also, neural networks may be involved in the activation of the AVP- and OXT-producing cells in the SON and PVN. It is worth noting that the expression level of c-fos mRNA and the number of Fos-LI in the PVN were greater than those in the SON after i.c.v. administration of NPW30 (in each dose; Fig. 2, 7, and 8). One possibility why the PVN cells were more activated than the SON cells is that the data obtained from the PVN may reflect the total changes of parvocellular cells and magnocellular cells, because the magnocellular and parvocellular divisions of the PVN were not considered for the measurement of the c-fos mRNA levels by quantitative imaging analysis. The other possibility is that the signal in the ependymal cells may contaminate the data obtained from the PVN, because the area of ependymal cells is close to the PVN. In addition, the activation of the c-fos gene and Fos expression in the PVN by NPW-administration may be more sensitive than that in the SON, because Levine et al.(2005) demonstrated in the Fos study, that the regional density of Fos-LI in the PVN was much higher than that of Fos-LI in the SON after i.c.v. administration of NPW. It is also worth noting that the c-fos gene was expressed throughout the ependymal cells of the third ventricle after i.c.v. administration of NPW30. The expression levels of the c-fos mRNA after i.c.v. administration of NPW30 were relatively stronger than those after i.c.v. administration of the vehicle. It may be possible that ependymal cells around the third ventricle may be activated by endogenous NPW30 in an autocrine and/or paracrine manner, because the NPW-LI ependymal cells and fibers were exhibited in lining the third ventricle of rats (Dun et al. 2003). However, as there are no previous reports regarding the presence of GPR7 in the ependymal cells, the possibility of a non-specific effect by i.c.v. administration of NPW30 cannot be excluded.
Previous studies also described that centrally administered NPW23 increased the release of corticosterone (Baker et al. 2003, Taylor et al. 2005). They suggested that centrally administered NPW activated the hypothalamo–pituitary–adrenal axis. We have demonstrated that c-fos mRNA and Fos-LI were detected throughout the SON and in the magnocellular and parvocellular divisions of the PVN after i.c.v. administration of NPW30. These results indicate that the release of corticosterone may be stimulated by both AVP and CRH.
The present study also showed that i.c.v. administration of NPW30 caused a rapid increase in the expression of AVP hnRNA in the SON and PVN. As the analysis of AVP hnRNA using intronic in situ hybridization histochemistry can detect a significant change in AVP gene transcription in the SON and PVN after stress, such as osmotic change, AVP hnRNA is a sensitive indicator for AVP gene transcription (Herman et al. 1991, Priou et al. 1993, Arima et al. 1999, Kawasaki et al. 2005). In this study, the elevation in AVP hnRNA levels is consistent with the result of elevated plasma AVP after i.c.v. administration of NPW30. According to the representative dark- and bright-field photomicrographs (Fig. 10), the expressing cells of AVP hnRNA were observed throughout the SON and in the magnocellular and parvocellular divisions of the PVN. Several studies demonstrated that AVP hnRNA levels ware rapidly and sensitively increased in response to acute stress (Kovacs & Sawchenko 1996, Ma et al. 1997a, 1997b). Thus, NPW, which induces AVP gene expression, may be involved in stress-related responses in the CNS.
In conclusion, centrally administered NPW30 activates AVP- and OXT-producing MNCs and stimulates secretion of AVP and OXT in conscious rats.
Time-course effects of i.c.v. administration of neuropeptide W-30 (NPW30; 1.4 and 2.8 nmol/rat) or 0.9% saline (vehicle) on plasma concentrations of (A) arginine vasopressin (AVP) and (B) oxytocin (OXT) in conscious rats. Data for plasma concentrations of AVP and OXT are expressed as means±S.E.M. (each group, n=6). *P<0.01, compared with vehicle-administered rats. †P<0.05, ‡P<0.01, compared with NPW (1.4 nmol/rat)-administered rats.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Dose–response effects of i.c.v. administration of NPW30 (0.3, 1.4, and 2.8 nmol/rat) or vehicle on c-fos transcript prevalence in the (A) SON and (B) PVN. Values represent means±S.E.M. (each group, n=6). *P<0.05, †P<0.01, compared with vehicle-administered rats.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Time-course effects of i.c.v. administration of NPW30 (1.4 nmol/rat) or vehicle on c-fos transcript prevalence in (A) SON and (B) PVN. Values represent means±S.E.M. (each group, n=6). *P<0.01, compared with vehicle-administered rats.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Representative dark-field photomicrographs (A)–(D) of emulsion-dipped slides hybridized to 35S-labeled oligodeoxy-nucleotide probe complementary to c-fos mRNA in the SON (A) and (C) and PVN (B) and (D) 30 min after i.c.v. administration of NPW30 (1.4 nmol/rat) (C) and (D) or vehicle (A) and (B). After development, these sections (C) and (D) were stained by cresylecht violet as representative bright-field photomicrographs (E) and (F). Bars=100 μm. 3V, third ventricle; OX, optic chiasma.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Coexistence of Fos-like immunoreactivity (LI) and AVP-/ OXT-LI in the SON 90 min after i.c.v. administration of NPW30 (2.8 nmol/rat). (A) Coexistence of Fos-LI (brown) and AVP-LI (violet). (B) Enlargement from the boxed areas in (A). (C) Coexistence of Fos-LI (brown) and OXT-LI (violet). (D) Enlargement from the boxed areas in (C). Black arrowheads indicate coexistence of nuclear Fos-LI and AVP-/OXT-LI. White arrowhead indicates AVP-LI without Fos-LI. Bars=100 μm. OX, optic chiasma.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Coexistence of Fos-LI and AVP-/OXT-LI in the PVN 90 min after i.c.v. administration of NPW30 (2.8 nmol/rat). (A) Coexistence of Fos-LI (brown) and AVP-LI (violet). (B) Enlargement from the boxed areas in (A). (C) Coexistence of Fos-LI (brown) and OXT-LI (violet). (D) Enlargement from the boxed areas in (C). Black arrowheads indicate coexistence of nuclear Fos-LI and AVP-/OXT-LI. White arrowhead indicates AVP-LI without Fos-LI. Bars= 100 μm. 3V, third ventricle.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Topographical mapping of Fos-LI and AVP-/OXT-LI in the SON 90 min after i.c.v. administration of vehicle (A) and (B) or NPW30 (2.8 nmol/rat) (C) and (D). (A) and (C) Coexistence of Fos-and AVP-LI (closed square). (C) and (D) Coexistence of Fos- and OXT-LI (closed triangle). In each panel, three 30 μm thick coronal sections from the anterior to posterior SON were selected. The open circle indicates a Fos-LI-positive cell, the open square indicates an AVP-LI-positive cell, and the closed square, a cell immunoreactive for both Fos and AVP. The open triangle indicates an OXT-LI-positive cell, and the closed triangle, a cell immunoreactive for both Fos and OXT. Bar indicates 100 μm. OX, optic chiasma.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Topographical mapping of Fos-LI and AVP-/OXT-LI in the PVN 90 min after i.c.v. administration of vehicle (A) and (B) or NPW30 (2.8 nmol/rat) (C) and (D). (A) and (C) Coexistence of Fos-LI and AVP-LI (closed square). (C) and (D) Coexistence of Fos-LI and OXT-LI (closed triangle). In each panel, two 30 μm thick coronal sections from the anterior to posterior PVN were selected. The open circle indicates a Fos-LI-positive cell, the open square, an AVP-LI-positive cell, and the closed square, a cell immunoreactive for both Fos and AVP. The open triangle indicates an OXT-LI-positive cell, and the closed triangle, a cell immunoreactive for both Fos and OXT. Bar indicates 100 μm. 3V, third ventricle.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Time-course effects of i.c.v. administration of NPW30 (1.4 nmol/rat) or vehicle on AVP heteronuclear (hn)RNA transcript prevalence in (A) SON and (B) PVN. Values represent the means±S.E.M. (each group, n=6). *P<0.01, compared with vehicle-administered rats.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
Representative dark-field photomicrographs of emulsion-dipped slides hybridized to 35S-labeled oligodeoxynucleotide probe complementary to AVP hnRNA in SON (A) and (C), and PVN (B) and (D), 30 min after i.c.v. administration of NPW30 (1.4 nmol/rat) (C) and (D) or vehicle (A) and (B). After development, these sections (C) and (D) were stained by cresylecht violet as representative bright-field photomicrographs (E) and (F). Bars=100 μm. 3V, third ventricle; OX, optic chiasma.
Citation: Journal of Endocrinology 190, 2; 10.1677/joe.1.06636
We thank Akio Takeuchi for his technical assistance. This paper was supported in part by UOEH Grant for Advanced Research, Grant-in-Aid for Science Research (B), no. 16390061 for Y U and Grant-in-Aid for Science Research (C), no. 16591518 for H O from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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