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Distribution of neuropeptide W in the rat brain
Neuropeptides 44 (2010) 99–106
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Neuropeptides journal homepage: www.elsevier.com/locate/npep
Special Issue on Peptide Receptors: Focus on Neuropeptides and Kinins
Distribution of neuropeptide W in the rat brain Fumiko Takenoya a,b, Michiko Yagi a, Haruaki Kageyama a, Kanako Shiba a, Kei Endo a, Naoko Nonaka c, Yukari Date d, Masamitsu Nakazato e, Seiji Shioda a,* a
Department of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Physical Education, Hoshi University School of Pharmacy and Pharmaceutical Science, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan c Department of Oral Anatomy and Developmental Biology, School of Dentistry, Showa University, 1-5-8 Hatanodai Shinagawa-ku, Tokyo 142-8555, Japan d Frontier Science Research Center, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan e Division of Neurology, Respirology, Endocrinology, and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan b
a r t i c l e
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Article history: Available online 30 November 2009 Keywords: Neuropeptide W Hypothalamus Distribution Immunohistochemistry Rat
a b s t r a c t Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction O’Dowd et al. (1995) cloned two novel genes, GPR7 (NPBWR1) and GPR8 (NPWBR2), that encode opioid- and somatostatin-like
orphan G-protein-coupled receptors (GPCRs) in the brain. NPBWR1 and NPBWR2 share 70% nucleotide and 64% amino acid identities with each other, with NPBWR1 being found in both humans and rodents whereas NPBWR2 is apparently expressed only in humans
Abbreviations: ac, Aterior commissure; aca, Aterior commissure, anterior part; acp, Aterior commissure, posterior part acp; AP, Area postrema; Aq, Aqueduct; Arc, Arcuate hypothalamic nucleus; ArcL, Arcuate nucleus, lateral part ArcL; BSTLP, Bed nucleus of the stria terminalis, lateral division, posterior part; BSTMA, Bed nucleus of the stria terminalis, medial division, anterior part; BSTMV, Bed nucleus of the stria terminalis, medial division, ventral part; BSTMPL, Bed nucleus of the stria terminalis, medial division, posterolateral part; Ce, Central amygdaloid nucleus; CeC, Central amygdaloid nucleus, capsular part; CeL, Central amygdaloid nucleus, lateral division; CeM, Central amygdaloid nucleus, medial division; cp, Cerebral peduncle, basal part; DLPAG, Ddorsolateral periaqueductal gray; DTM, Dorsal tuberomammillary nucleus; DRC, Dorsal raphe nucleus, caudal part; ec, External capsule; EW, Edinger–Westphal nucleus; f, Fornix; HDB, Nucleus of the horizontal limb of the diagonal band; ic, Internal capsule; ICj, Islands of Calleja; IPR, Interpeduncular nucleus, rostral subnucleus; LA, Lateroanterior hypothalamic nucleus; LGP, Lateral globus pallidus; LH, Lateral hypothalamic area; LPAG, Ventrolateral periaqueductal gray; LSV, Lateral septal nucleus, ventral part; Ml, Medial lemniscus; LDTg, Laterodorsal tegmental nucleus; MnR, Median raphe nucleus; MPA, Medial preoptic area; MPO, Medial preoptic nucleus; MPOC, Medial preoptic nucleus, central part; MRe, Mammillary recess of the 3rd ventricle; ox, Optic chiasm; opt, Optic tract; OPT, Olivary pretectal nucleus; PaAP, Paraventricular hypothalamic nucleus, anterior parvicellular part; PaMP, Paraventricular hypothalamic nucleus, medial parvicellular part; PaPo, Paraventricular hypothalamic nucleus, posterior part; PaV, Paraventricular hypothalamic nucleus, ventral part; Pe, Periventricular hypothalamic nucleus; PMD, Premammillary nucleus, dorsal part; RMg, Raphe magnus nucleus; Py, Pyramidal tract; pr, Prepositus nucleus; scp, Superior cerebellar peduncle; Sch, Suprachiasmatic nucleus; SO, Supraoptic nucleus; sox, Supraoptic decussation; st, Stria terminalis; VLPAG, Ventrolateral periaqueductal gray; VMPO, Ventromedial preoptic nucleus; VMHA, Ventromedial hypothalamic nucleus, anterior part; VTA, Ventral tegmental area; VP, Ventral pallidum; 7, Accessory abducens/facial nucleus; 12, Hypoglossal nucleus. * Corresponding author. Tel.: +81 3 3784 8103; fax: +81 3 3784 6815. E-mail address: [email protected] (S. Shioda). 0143-4179/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2009.10.007
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(Lee et al., 1999). NPBWR1 and NPBWR2 mRNA has been detected at high levels in tissues of the human central nervous system (CNS) using RT-PCR analysis (Fujii et al., 2002). In particular, high levels of NPBWR1 mRNA were found in the hippocampus and amygdala (Brezillon et al., 2003) whereas, in the rat, strong NPBWR1 mRNA expression was detected in the hypothalamus and amygdala (Fujii et al., 2002). Similarly, in situ hybridization studies have revealed that NPBWR1 mRNA is present in the rat hypothalamus, including the arcuate nucleus (ARC), ventromedial nucleus (VMH), paraventricular nucleus (PVN), dorsomedial nucleus (DMH) and supraoptic nucleus (SON) (Lee et al., 1999), which are well known for their role in feeding regulation and energy homeostasis. Recently, several studies have reported the identification of two endogenous ligands for NPBWR1 and NPBWR2, neuropeptides W and B (NPW and NPB, respectively) (Fujii et al., 2002; Brezillon et al., 2003; Tanaka et al., 2003), which were isolated from the porcine hypothalamus. In the case of NPW, this has led to the characterization of two endogenous peptides consisting of 23- and 30-amino acid residues, termed neuropeptide W-23 (NPW23) and neuropeptide W30 (NPW30), respectively. Synthetic NPW23 and NPW30 have been shown to be similarly effective in binding and activating both NPBWR1 and NPBWR2 (Shimomura et al., 2002; Tanaka et al., 2003). In in vitro experiments, human NPW23 and NPW30 were shown to have a similar potency in terms of activating human NPBWR1. In rat, NPBWR1 is expressed in the hypothalamus, including its feeding centers, and administration of NPW to NPBWR1 knockout mice results in hyperphagic and decreased energy expenditure effects, suggesting a modulatory role for NPW in the control of feeding regulation. Recently, a physiological study reported that the intracerebroventricular (icv) injection of NPW induced acute food intake for the first 2 h in the light phase (Shimomura et al., 2002). However, Mondal et al. (Mondal et al. (2003)) reported that icv infusion of NPW significantly reduced dark-phase feeding 4 h after administration, an effect that was maintained for 48 h at higher doses, concomitant with increased body temperature and heat production. Administration of NPW30 has also been shown to increase arterial blood pressure heart rate and plasma catecholamine levels in rats (Yu et al., 2007), with Baker et al. demonstrating that administration of NPW23 elevates prolactin, corticosterone (Baker et al., 2003), and growth hormone levels. In addition, Niimi and Murao (Niimi and Murao (2005)) have reported that icv administration of NPW results in significant Fos expression in the PVN, suggesting that NPW is involved in stress-responsive signal transduction, and may be a modulator of the hypothalamus–pituitary–adrenal axis. On the other hand, the presence of NPBWR1 in the PVN is suggestive of a role in the modulation of neuroendocrine functions. Finally, Yamamoto et al. (Yamamoto et al. (2005)) have demonstrated that intrathecal administration of NPW23 or NPB in rats also suppresses inflammatory pain. Overall, these various studies reveal that NPW is a multifunctional peptide that mediates a range of physiological outcomes. Based on immunohistochemical analysis, NPW mRNA expression in the human CNS has been shown to be strongest in the substantia nigra, amygdala and hippocampus (Fujii et al., 2002; Singh et al., 2004). We (Takenoya et al., 2008), together with Dun et al. (Dun et al. (2003)) have also reported that NPW-like immunoreactive (NPW-LI) cell bodies are found in various regions of the rat brain, including the preoptic area (POA), PVN, SON, ARC, and dorsal and lateral hypothalamic areas, as well as the anterior and posterior pituitary gland. In contrast, Kitamura et al. (Kitamura et al. (2006)) reported expression in the Edinger–Westphal nucleus (EW), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and medial parabrachial nucleus (MPB), but saw no NPWLI cell bodies in the PVN. Given these somewhat conflicting results, the present study was designed to provide a detailed analysis of
NPW expression in the rat brain. This was achieved by first determining NPW gene expression by reverse-transcription polymerase chain reaction (RT-PCR), followed by immunohistochemical localization of NPW-LI neurons and the neuronal network between NPW-containing neurons and other hypothalamic neurons at the light and electron microscopic levels. 2. Materials and methods 2.1. Animals Male Wistar and Sprague–Dawley (SD) rats (Saitama Experimental Animal Supply, Saitama, Japan) weighing approximately 300 g were used. The animals were maintained on a 12/12 h light/dark cycle and supplied with standard laboratory chow and tap water ad libitum. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Showa University. Rats were placed under deep Nembutal anesthesia (40 mg/ kg, i.p.) and colchicine (200 lg/25 ll saline) injected into the third ventricle of the brain. After 48 h, the rats were perfused through the ascending aorta with 50 ml of saline (37 °C), followed by 250–300 ml of 2% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) fixative for 20 min. The brains were enucleated, trimmed, and immersed in the same fixative for 12 h at 48 °C. After washing, the fixed brains were transferred to a solution containing 20% sucrose in 0.1 M PB for 2 days at 4 °C. 2.2. RT-PCR The hypothalamic LH, ARC, VMH and PVN were punched out of the brain slices of the SD rats. The stomach was also taken from the SD rat. Total RNA was prepared from these tissues using TRIzol reagent (Invitrogen Corp), according to the manufacturer’s protocol. Total RNA from the stomach was used as a positive control for NPW (Mondal et al., 2003, 2006, 2008). Total RNA was treated with DNase (Roche, Basel, Switzerland). RNA was quantified spectrophotometrically and confirmed by ethidium bromide staining of 18S and 28S ribosomal RNA after electrophoresis on 1.0% agarose/formaldehyde gel. Four micrograms of total RNA were converted into cDNA using oligo d(T)12–18 primer (Invitrogen Corp.) and SuperScript III (Invitrogen Corp.) in a RT-reaction mixture (20 ll). PCR was performed using 0.8 ll of the RT-reaction mixture, 0.5 lM of each primer and AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster, CA, USA) in a total reaction volume of 25 ll. The primers used for rat NPW were 50 -GAGCTGTGGGAGGTA CGAAG-30 and 50 -CTGACAGGATCGGCAAAGAT-30 (GeneBank Accession No. AB084278). The PCR products were visualized by ethidium bromide staining under UV light following electrophoresis on a 1.5% agarose gel. After TA-cloning of PCR products, the nucleotide sequence of each inserted cDNA in the plasmid was confirmed by an automatic sequencing analyzer (ABI377, Applied Biosystems). 2.3. Preparation of sections Rats were anesthetized with pentobarbital (50 mg/kg, Dainippon Pharmaceutical, Saitama, Japan) then perfused with saline, followed by 250–300 ml of 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was transferred to 2% PFA/PB overnight at 4 °C, after which it was transferred first to 0.1 M PB solution containing 20% sucrose at 4 °C and then to 0.1 M PB solution containing 30% sucrose at 4 °C. The fixed brain was finally embedded in O.C.T compound in liquid nitrogen-cooled isopentane and stored at 80 °C. Sections 7 lm-thick were subsequently cut by cryostat (MICROM HM 500; MICROM, Heidelberg, Germany), and used for immunohistochemistry.
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2.4. Single staining
3.2. Localization of NPW-positive cell bodies and nerve fibers in brain
Sections were blocked in phosphate-buffered saline (PBS) containing 10% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 60 min. Then, in order to detect NPW immunoreactivity, they were incubated overnight at 4 °C with anti-NPW antiserum (1:4000), (Mondal et al., 2003, 2006, 2008) washed three times with PBS for 5 min, and incubated with biotinylated anti-mouse IgG (1:400; DAKO, Carpinteria, CA, USA) for 2 h at room temperature (RT). The signals were amplified by ABC kit (Vector Laboratories, Inc.), and visualized using a peroxidase substrate (diaminobenzidine; DAB) kit (Vector Laboratories, Inc.) following washes with PBS. After dehydration, the mounted sections were coverslipped with malinol (Muto Pure Chemicals Ltd., Tokyo, Japan). NPW-like immunoreactivity (NPW-LI) was detected with the optical microscope (PROVIS, Olympus, Tokyo, Japan). As a control, the procedure described above was also performed omitting the primary antibody. Some frozen and vibratome sections were used as controls in which no anti-NPW antiserum or NPW-adsorbed NPW antiserum was used in the incubation process. No specific immunoreactivity was observed in these control sections.
NPW-LI cell bodies were observed in many regions of the rat brain (Figs. 2–4). NPW-positive cell bodies were seen in the islands of Calleja (ICj) (Fig. 2A and B), the ventrolateral hypothalamic nucleus (VLH) (Fig. 2D), the anterior parvocellular section of the paraventricular hypothalamic nucleus (PaAP) (Fig. 2E), the medial parvocellular section of the paraventricular hypothalamic nucleus (PaMP) (Fig. 2H), the posterior paraventricular hypothalamic nucleus (PaPe) (Fig. 2I), the lateral division of the posterior bed nucleus of the stria terminalis, (BSTLP) (Fig. 2F), the dorsal section of the dorsomedial hypothalamic nucleus (DMD) (Fig. 2K and L), the VMH (Fig. 3A and B), the ARC (Fig. 3C and D), LH (Fig. 3C and E), the dorsal premammillary nucleus (PDM) (Fig. 3F), the PAG (Fig. 3G), the EW (Fig. 3H), the central part of the lateral parabrachial nucleus (LPBC), the medial parabrachial nucleus (MPB) (Fig. 3I), the prepositus nucleus (Pr) (Fig. 3K), and the raphe nucleus (Fig. 3L). In addition, NPW-positive axon terminals were found in the lateral septal nucleus (LS) (Fig. 2C), the ventral part of the lateral division of the bed nucleus of the stria terminalis (BSTLV) (Fig. 2G), the amygdala (Fig. 2J), the LH (Fig. 2K and M), lateral parabrachial nucleus (LPB) (Fig. 3I) and the lateral PAG (LPAG) (Fig. 3J).
2.5. Electron microscopy
3.3. Electron microscopic observation
For electron microscopy, vibratome sections (40 lm thickness) that had been fixed as described above were first incubated in normal horse serum (1:20) for 20 min, and then in mouse anti-NPW antiserum (diluted to 1:1000) for 2 h at RT, followed by overnight incubation at 4 °C. On the following day, the sections were incubated with biotinylated anti-mouse IgG (DAKO, Carpinteria, CA, USA) for 1 h and then with ABC for 45 min at (Vectastain Elite ABC kit, Vector Laboratories). Following this, they were treated with DAB in 0.05 M Tris–HCl (pH 7.6) buffer including 0.005% hydrogen peroxide for about 3 min in the dark. After the DAB reaction, some of the sections were further treated with silver–gold intensification. These sections were post-fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 1 h at 4 °C, dehydrated in a graded ethanol series, and then embedded in a mixture of Epon–Araldite. Ultrathin sections were cut and examined by using a Hitachi H-7000 electron microscope. The specificity of the NPW antiserum used in this study has been reported elsewhere (Mondal et al., 2006).
At the electron microscopic level, many NPW-LI neurons were identified in the PVN (Fig. 5A). NPW-LI cell bodies often received inputs from other unknown neurons in the PVN (Fig. 5B), and NPW-LI was also demonstrated in dense-core vesicles (Fig. 5C). NPW-LI cell bodies often made synaptic contact with unknown axon terminals in the PVN (Fig. 5D). NPW-positive axon terminals were found to make synaptic contacts with unknown dendritic processes in the PVN (Fig. 5E), as well as the VMH (Fig. 5F). In the amygdala, NPW-like immunoreactivity was seen in dense-core vesicles (Fig 5G) and NPW-LI dendritic processes often received synaptic inputs from other unknown axon terminals (Fig. 5H).
3. Results 3.1. RT-PCR RT-PCR analysis was used to determine NPW mRNA expression in the rat hypothalamus. This revealed a PCR product of NPW corresponding to the predicted size of 211 bp (Fig. 1). NPW mRNA was expressed in the PVN, LH, ARC and VMH (Fig. 1). Expression levels of its mRNA in the PVN, LH, ARC and VMH were lower than that in the whole hypothalamus.
Fig. 1. RT-PCR analysis of NPW mRNA expression in the rat hypothalamic nuclei. RT-PCR analysis showed a PCR product corresponding to the predicted size of 211 bp.
4. Discussion In our present study, we observed distribution and localization of NPW-LI in rat brain but there is no clear species difference between Wistar and SD rats. There have been published two morphological study of NPW in brain by use of immunohistochemistry (Dun et al., 2003 and Kitamura et al., 2006). There are several different data on the distribution and localization of NPW-LI in brain tissues. Based on our present observation it may be caused from the different sources of the antibodies but not from the species difference. The present study is the first to demonstrate, based on RT-PCR analysis, that NPW mRNA is expressed in the PVN, LH, ARC, and VMH of the rat hypothalamus. In addition, we immunohistochemically examined the distribution of NPW-LI in rat brain pretreated with colchicine, a process which has recently been reported to dramatically enhance such immunoreactivity (Takenoya et al., 2008). Light microscopic observation revealed NPW-immunoreactive cell bodies in a number of areas, including the hypothalamus, midbrain, pons, and medulla oblongata. Many NPW-containing fibers were found in the LS, BST, amygdala, LH, PMD, PAG, and Pr. These results are schematically summarized in Fig. 4. We first identified at the electron microscopic level the presence of NPW-LI cell bodies in the PVN, VMH, and amygdala. These neurons were shown to make synaptic contact with unknown axon terminals in the PVN and ARC. A wide distribution of NPW-LI neurons in the brain suggests a role for NPW in feeding regulation as well as many physiological functions.
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Fig. 2. Photomicrographs of sections through colchicine-treated rat brain labeled with anti-NPW antiserum. NPW-positive cell bodies were seen in the islands of Calleja (ICj) (A and B), the ventrolateral hypothalamic nucleus (VLH) (D), the anterior parvocellular section of the paraventricular hypothalamic nucleus (PaAP) (E), the posterior part of the lateral division of the bed nucleus of the stria terminalis (BSTLP) (F), the medial parvocellular section of the paraventricular hypothalamic nucleus (PaMP) (H), the posterior paraventricular hypothalamic nucleus (PaPo) (I), the dorsal section of the dorsomedial hypothalamic nucleus (DMD) (K and L). NPW-positive axon terminals were seen in the lateral septal nucleus (LS) (C), the ventral part of the lateral division of the bed nucleus of the stria terminalis (BSTLV) (G), the amygdala (J), the LH (K and M). Scale bar = 250 lm (K), 100 lm (A, C, and F–I), 50 lm (B, D, E, J, L, and M). 3 V: 3rd ventricle.
Recently, it has been reported, based on in situ hybridization study, that NPW mRNA is expressed in the PAG, EW, and ventral tegmental area in the rat brain, but not in the PVN in mice and rats (Kitamura et al., 2006). However, in the present study, we identified NPW mRNA expression in the PVN as well as in the ARC, LH and VMH based on RT-PCR analysis. One plausible explanation for this discrepancy is that since the expression level of its mRNA is very low in the PVN, ARC, LH and VMH, it is difficult to detect NPW mRNA using in situ hybridization without signal amplification method such as tyramide signal amplification (TSA) system (Seki et al., 2008). NPBWR1 mRNA has previously been reported in the VMH (Williams et al., 2001), as well as the DMH, PVN, and ARC (Lee et al.,
1999). The abundance of NPBWR1 mRNA seen in the hypothalamus suggests that it may be involved in feeding regulation and metabolic disorders. In previous studies, icv administration of NPW suppresses food intake in the dark phase (Mondal et al., 2003). By contrast, during the light phase, NPW administration has been shown to stimulate feeding (Shimomura et al., 2002; Baker et al., 2003). We hypothesize that there are many NPW-mediated neural pathways from the ARC, VMH, and PVN to other brain areas, as a result of which NPW is able to act in concert with other neuropeptides to modulate the regulation of feeding behavior. A very few feeding-regulating peptides are isolated from the VMH (Sternson et al., 2005). Interestingly, in this study, we identified NPW-LI cell bodies in the VMH, a satiety center. Leptin acts on
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Fig. 3. Photomicrographs of sections through colchicine-treated rat brain labeled with anti-NPW antiserum. NPW-positive cell bodies were seen in the ventromedial hypothalamic nucleus (VMH) (A and B), the arcuate nucleus (ARC) (C and D), the lateral hypothalamus (LH) (C and E), the dorsal premammillary nucleus (PMD) (F), the periaqueductal gray (PAG) (G), the Edinger–Westphal nucleus (EW) (H), the central lateral parabrachial nucleus (LPBC) (I), the medial parabrachial nucleus (MPB) (I), the prepositus nucleus (Pr) (K), the raphe magnus nucleus (RMg) (L). NPW-positive axon terminals were seen in the lateral PAG (LPAG) (J), and the prepositus nucleus (Pr) (K). Scale bar = 250 lm (C), 100 lm (A, D, F, H, and J–L), 50 lm (B, E, G, and I). 3 V: 3rd ventricle. 4 V: 4th ventricle.
the VMH where its receptors are abundantly expressed, and reduces food intake. Changes of synaptic activity of projection from the VMH to the proopiomelanocortin (POMC) neurons in the ARC are known to inhibit feeding behavior (Elmquist et al., 1997; Koyama et al., 1998) and to regulate glucose level and energy homeostasis (Schwartz et al., 2000). Our present findings suggest that leptin acts on NPW-containing neurons in the VMH, and NPW may reduce food intake via POMC neuronal pathway. Further studies are required to identify neuronal interactions between NPW and POMC or leptin. The findings of the present study are similar to those reported by Dun et al. (Dun et al. (2003)), who reported NPW-LI cells in the PVH, supraoptic nucleus (SON), dorsal hypothalamus, LH, per-
ifornical nucleus, ARC, and posterior pituitary. It has also been reported that icv injection of NPW induced c-Fos expression in the rat hypothalamic areas such as the SON and PVN (Kawasaki et al., 2006). Injection of NPW at high dose into the LH increased food intake during 4 h after injection (Kawasaki et al., 2006). In contrast to the result obtained following NPW injection into the PVN, there was no effect during 24 h after injection (Levine et al., 2005). We have reported the first anatomical evidence of NPWcontaining efferent neuron population in the hypothalamus, and have shown that these NPW-containing neurons interact with orexin- and melanin-concentrating hormone-containing neurons in the LH (Takenoya et al., 2008). These data support the idea that feeding regulation is one of the important functions of NPW.
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Fig. 4. Schematic drawings showing the distribution of NPW-containing fibers (left) and cells (right) in the brain.
In the forebrain, we observed that the ICj receive inputs from the caudal part of the amygdala, which processes emotional memory (Ubeda-Banon et al., 2008) as well as from the septum, nucleus accumbens, and piriform cortex. In addition, the ICj also receives information from neurons in the substantia nigra and ventral tegmental area, located in the midbrain (Diaz et al., 1995).
We have found in this study that numerous NPW-LI cell bodies and fibers are observed in the POA and the BST which are involved in anxiety and the stress response. Similarly, the presence of NPBWR1 mRNA has been confirmed by in situ hybridization in the POA and BST (Tanaka et al., 2003). It is well known that NPW-containing neurons in the midbrain project to the central
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Fig. 5. Electron micrographs showing the NPW. (A) NPW-LI neurons were identified in the PVN. N, nucleus. (B) NPW-LI cell bodies are often received inputs from other unknown neurons (arrowhead) in the PVN. N, nucleus. (C) NPW-LI is demonstrated in dense-core vesicles as shown arrows. (D) NPW-LI cell bodies are makes synaptic contact (arrowhead) with unknown axon terminals in the PVN. (E) NPW-positive axon terminals (asterisk) are makes synaptic contact (arrowhead) with unknown dendrites processes in the PVN. (F) NPW-positive axon terminals (asterisk) are makes synaptic contact (arrowhead) with unknown dendrites processes in the VMH. (G) In the amygdara NPW-LI is observed in dense-core vesicles as shown arrows. (H) NPW-LI dendritic processes were often received (arrowhead) inputs from other unknown neurons in the amygdara. Scale bars = 0.2 lm.
amygdala (CeA) and BST. It is suggested that NPW is a regulator for the emotive responses, fear and anxiety by modulating the CeA and BST neurons (Kitamura et al., 2006; Shimomura et al., 2002).
The PAG is a region known to play a role in the descending modulation of pain and in defensive behavior (Monassi et al., 1999), with inputs from the spinomesencephalic tract in addition to affer-
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ent fibers from the hypothalamus, amygdala and brainstem. In addition, we found many NPW-LI cell bodies in the LPB, around the superior cerebellar peduncle, which are involved in the integration of taste information and the associated autonomic response (Matsuo et al., 1984). Fibers from the LPB are shown to project the amygdala and dorsal hypothalamus (Li et al., 2005; Saggu and Lundy, 2008), with the spinal cord–LPB–amygdala/hypothalamus pathway being known to participate in pain, anxiety and the autonomic nervous response (Day et al., 2004). Therefore, our present study provides the background on the involvement of NPW in modulation of response to anxiety, inflammatory pain (Kelly et al., 2005; Yamamoto et al., 2005; Singh and Davenport, 2006; Hondo et al., 2008). The LH, amygdala and BST are implicated to regulate feeding regulation and taste. In particular, nerve fibers in the LH are shown to project to premotoneurons in the NST and LPB (Matsuo et al., 1984). These premotoneurons are well known to innervate the salivary glands and tongue. NPW-containing cell bodies were found to be present in the LH, BST and LPB, in addition, NPW-containing fibers are present in the LPB. The above findings suggest that NPW is involved not only in regulation of food intake but also in saliva secretion, chewing and tongue movements. Further studies are required to analyze the precisely the neuronal network underpinning to understand each of these physiological functions. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture (F.T., H.K., S.S.), and a Hightechnology Research Center Project from the Ministry of Education, Science, Sports and Culture of Japan (S.S.). References Baker, J.R., Cardinal, K., Bober, C., Taylor, M.M., Samson, W.K., 2003. Neuropeptide W acts in brain to control prolactin, corticosterone, and growth hormone release. Endocrinology 144, 2816–2821. Brezillon, S., Lannoy, V., Franssen, J.D., Le Poul, E., Dupriez, V., Lucchetti, J., Detheux, M., Parmentier, M., 2003. Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8. J. Biol. Chem. 278, 776–783. Day, H.E., Masini, C.V., Campeau, S., 2004. The pattern of brain c-Fos mRNA induced by a component of fox odor, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, suggests both systemic and processive stress characteristics. Brain Res. 1025, 139–151. Diaz, J., Levesque, D., Lammers, C.H., Griffon, N., Martres, M.P., Schwartz, J.C., Sokoloff, P., 1995. Phenotypical characterization of neurons expressing the dopamine D3 receptor in the rat brain. Neuroscience 65, 731–745. Dun, S.L., Brailoiu, G.C., Yang, J., Chang, J.K., Dun, N.J., 2003. Neuropeptide Wimmunoreactivity in the hypothalamus and pituitary of the rat. Neurosci. Lett. 349, 71–74. Elmquist, J.K., Ahima, R.S., Maratos-Flier, E., Flier, J.S., Saper, C.B., 1997. Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138, 839–842. Fujii, R., Yoshida, H., Fukusumi, S., Habata, Y., Hosoya, M., Kawamata, Y., Yano, T., Hinuma, S., Kitada, C., Asami, T., Mori, M., Fujisawa, Y., Fujino, M., 2002. Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7. J. Biol. Chem. 277, 34010–34016. Hondo, M., Ishii, M., Sakurai, T., 2008. The NPB/NPW neuropeptide system and its role in regulating energy homeostasis, pain, and emotion. Results Probl. Cell Differ. 46, 239–256. Kawasaki, M., Onaka, T., Nakazato, M., Saito, J., Mera, T., Hashimoto, H., Fujihara, H., Okimoto, N., Ohnishi, H., Nakamura, T., Ueta, Y., 2006. Centrally administered neuropeptide W-30 activates magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei with neurosecretion in rats. J. Endocrinol. 190, 213–223. Kelly, M.A., Beuckmann, C.T., Williams, S.C., Sinton, C.M., Motoike, T., Richardson, J.A., Hammer, R.E., Garry, M.G., Yanagisawa, M., 2005. Neuropeptide B-deficient mice demonstrate hyperalgesia in response to inflammatory pain. Proc Natl Acad Sci USA 102, 9942–9947. Kitamura, Y., Tanaka, H., Motoike, T., Ishii, M., Williams, S.C., Yanagisawa, M., Sakurai, T., 2006. Distribution of neuropeptide W immunoreactivity and mRNA in adult rat brain. Brain Res. 1093, 123–134.
Koyama, K., Shimabukuro, M., Chen, G., Wang, M.Y., Lee, Y., Kalra, P.S., Dube, M.G., Kalra, S.P., Newgard, C.B., Unger, R.H., 1998. Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors. J. Clin. Invest. 102, 728–733. Lee, D.K., Nguyen, T., Porter, C.A., Cheng, R., George, S.R., O’Dowd, B.F., 1999. Two related G protein-coupled receptors: the distribution of GPR7 in rat brain and the absence of GPR8 in rodents. Brain Res. Mol. Brain Res. 71, 96–103. Levine, A.S., Winsky-Sommerer, R., Huitron-Resendiz, S., Grace, M.K., de Lecea, L., 2005. Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake. Am. J. Physiol. Regul. Int. Comp. Physiol. 288, R1727–1732. Li, C.S., Cho, Y.K., Smith, D.V., 2005. Modulation of parabrachial taste neurons by electrical and chemical stimulation of the lateral hypothalamus and amygdala. J. Neurophysiol. 93, 1183–1196. Matsuo, R., Shimizu, N., Kusano, K., 1984. Lateral hypothalamic modulation of oral sensory afferent activity in nucleus tractus solitarius neurons of rats. J. Neurosci. 4, 1201–1207. Monassi, C.R., Leite-Panissi, C.R., Menescal-de-Oliveira, L., 1999. Ventrolateral periaqueductal gray matter and the control of tonic immobility. Brain Res. Bull. 50, 201–208. Mondal, M.S., Yamaguchi, H., Date, Y., Shimbara, T., Toshinai, K., Shimomura, Y., Mori, M., Nakazato, M., 2003. A role for neuropeptide W in the regulation of feeding behavior. Endocrinology 144, 4729–4733 (Epub 2003 Aug, 4727). Mondal, M.S., Yamaguchi, H., Date, Y., Toshinai, K., Kawagoe, T., Tsuruta, T., Kageyama, H., Kawamura, Y., Shioda, S., Shimomura, Y., Mori, M., Nakazato, M., 2006. Neuropeptide W is present in antral G cells of rat, mouse, and human stomach. J. Endocrinol. 188, 49–57. Mondal, M.S., Yamaguchi, H., Date, Y., Tsuruta, T., Shimbara, T., Toshinai, K., Shimomura, Y., Mori, M., Nakazato, M., 2008. Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach. Regul. Pept. 145, 141–146. Niimi, M., Murao, K., 2005. Neuropeptide W as a stress mediator in the hypothalamus. Endocrine 27, 51–54. O’Dowd, B.F., Scheideler, M.A., Nguyen, T., Cheng, R., Rasmussen, J.S., Marchese, A., Zastawny, R., Heng, H.H., Tsui, L.C., Shi, X., et al., 1995. The cloning and chromosomal mapping of two novel human opioid–somatostatin-like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain. Genomics 28, 84–91. Saggu, S., Lundy, R.F., 2008. Forebrain neurons that project to the gustatory parabrachial nucleus in rat lack glutamic acid decarboxylase. Am. J. Physiol. Regul. Int. Comp. Physiol. 294, R52–57. Schwartz, M.W., Woods, S.C., Porte Jr., D., Seeley, R.J., Baskin, D.G., 2000. Central nervous system control of food intake. Nature 404, 661–671. Seki, M., Kageyama, H., Takenoya, F., Hirayama, M., Kintaka, Y., Inoue, S., Matsuno, R., Itabashi, K., Date, Y., Nakazato, M., Shioda, S., 2008. Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla. Regul. Pept. 145, 147–152. Shimomura, Y., Harada, M., Goto, M., Sugo, T., Matsumoto, Y., Abe, M., Watanabe, T., Asami, T., Kitada, C., Mori, M., Onda, H., Fujino, M., 2002. Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8. J. Biol. Chem. 277, 35826–35832. Singh, G., Davenport, A.P., 2006. Neuropeptide B and W: neurotransmitters in an emerging G-protein-coupled receptor system. Br. J. Pharmacol. 148, 1033– 1041. Singh, G., Maguire, J.J., Kuc, R.E., Fidock, M., Davenport, A.P., 2004. Identification and cellular localisation of NPW1 (GPR7) receptors for the novel neuropeptide W-23 by [125I]-NPW radioligand binding and immunocytochemistry. Brain Res. 1017, 222–226. Sternson, S.M., Shepherd, G.M., Friedman, J.M., 2005. Topographic mapping of VMH ? arcuate nucleus microcircuits and their reorganization by fasting. Nat. Neurosci. 8, 1356–1363. Takenoya, F., Kitamura, S., Kageyama, H., Nonaka, N., Seki, M., Itabashi, K., Date, Y., Nakazato, M., Shioda, S., 2008. Neuronal interactions between neuropeptide Wand orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus. Regul. Pept. 145, 159–164. Tanaka, H., Yoshida, T., Miyamoto, N., Motoike, T., Kurosu, H., Shibata, K., Yamanaka, A., Williams, S.C., Richardson, J.A., Tsujino, N., Garry, M.G., Lerner, M.R., King, D.S., O’Dowd, B.F., Sakurai, T., Yanagisawa, M., 2003. Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proc. Natl. Acad. Sci. USA 100, 6251–6256. Ubeda-Banon, I., Novejarque, A., Mohedano-Moriano, A., Pro-Sistiaga, P., Insausti, R., Martinez-Garcia, F., Lanuza, E., Martinez-Marcos, A., 2008. Vomeronasal inputs to the rodent ventral striatum. Brain Res. Bull. 75, 467–473. Williams, G., Bing, C., Cai, X.J., Harrold, J.A., King, P.J., Liu, X.H., 2001. The hypothalamus and the control of energy homeostasis: different circuits, different purposes. Physiol. Behav. 74, 683–701. Yamamoto, T., Saito, O., Shono, K., Tanabe, S., 2005. Anti-hyperalgesic effects of intrathecally administered neuropeptide W-23, and neuropeptide B, in tests of inflammatory pain in rats. Brain Res. 1045, 97–106. Yu, N., Chu, C., Kunitake, T., Kato, K., Nakazato, M., Kannan, H., 2007. Cardiovascular actions of central neuropeptide W in conscious rats. Regul. Pept. 138, 82–86.
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Neuropeptides journal homepage: www.elsevier.com/locate/npep
Special Issue on Peptide Receptors: Focus on Neuropeptides and Kinins
Distribution of neuropeptide W in the rat brain Fumiko Takenoya a,b, Michiko Yagi a, Haruaki Kageyama a, Kanako Shiba a, Kei Endo a, Naoko Nonaka c, Yukari Date d, Masamitsu Nakazato e, Seiji Shioda a,* a
Department of Anatomy, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Physical Education, Hoshi University School of Pharmacy and Pharmaceutical Science, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan c Department of Oral Anatomy and Developmental Biology, School of Dentistry, Showa University, 1-5-8 Hatanodai Shinagawa-ku, Tokyo 142-8555, Japan d Frontier Science Research Center, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan e Division of Neurology, Respirology, Endocrinology, and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan b
a r t i c l e
i n f o
Article history: Available online 30 November 2009 Keywords: Neuropeptide W Hypothalamus Distribution Immunohistochemistry Rat
a b s t r a c t Neuropeptide W (NPW), which was recently isolated from the porcine hypothalamus, has been identified as the endogenous ligand of the orphan G protein-coupled receptors GPR7 (NPBWR1) and GPR8 (NPBWR2). Infusion of NPW increases food intake in the light phase, whereas in the dark phase, it has the opposite effect. In this study, we used RT-PCR analysis to examine the gene expression of NPW mRNA in the rat brain, and performed a detailed analysis of the distribution of NPW-positive neurons by use of immunohistochemistry at both the light and electron microscopic levels. NPW mRNA expression was demonstrated in the hypothalamic paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMH) and lateral hypothalamus (LH). At the light microscopic level, NPW-like immunoreactive (NPW-LI) cell bodies were found in the preoptic area (POA), PVN, ARC, VMH, LH, PMD (dorsal premammillary nucleus), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and prepositus nucleus (Pr). NPW-LI axon terminals were shown in the POA, bed nucleus of the stria terminalis (BST), amygdala, PVN, ARC, VMH, LH, and PAG, LPB. In addition, at the electron microscopic level, NPW-LI cell bodies and dendritic processes were often seen to receive inputs from other unknown neurons in the ARC, PVN, VMH and amygdala. Our observations indicate that NPW-LI neurons widely distributed in the rat brain region. These finding suggest that NPW may have important roles in feeding behavior, energy homeostasis, emotional response and regulation of saliva secretion. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction O’Dowd et al. (1995) cloned two novel genes, GPR7 (NPBWR1) and GPR8 (NPWBR2), that encode opioid- and somatostatin-like
orphan G-protein-coupled receptors (GPCRs) in the brain. NPBWR1 and NPBWR2 share 70% nucleotide and 64% amino acid identities with each other, with NPBWR1 being found in both humans and rodents whereas NPBWR2 is apparently expressed only in humans
Abbreviations: ac, Aterior commissure; aca, Aterior commissure, anterior part; acp, Aterior commissure, posterior part acp; AP, Area postrema; Aq, Aqueduct; Arc, Arcuate hypothalamic nucleus; ArcL, Arcuate nucleus, lateral part ArcL; BSTLP, Bed nucleus of the stria terminalis, lateral division, posterior part; BSTMA, Bed nucleus of the stria terminalis, medial division, anterior part; BSTMV, Bed nucleus of the stria terminalis, medial division, ventral part; BSTMPL, Bed nucleus of the stria terminalis, medial division, posterolateral part; Ce, Central amygdaloid nucleus; CeC, Central amygdaloid nucleus, capsular part; CeL, Central amygdaloid nucleus, lateral division; CeM, Central amygdaloid nucleus, medial division; cp, Cerebral peduncle, basal part; DLPAG, Ddorsolateral periaqueductal gray; DTM, Dorsal tuberomammillary nucleus; DRC, Dorsal raphe nucleus, caudal part; ec, External capsule; EW, Edinger–Westphal nucleus; f, Fornix; HDB, Nucleus of the horizontal limb of the diagonal band; ic, Internal capsule; ICj, Islands of Calleja; IPR, Interpeduncular nucleus, rostral subnucleus; LA, Lateroanterior hypothalamic nucleus; LGP, Lateral globus pallidus; LH, Lateral hypothalamic area; LPAG, Ventrolateral periaqueductal gray; LSV, Lateral septal nucleus, ventral part; Ml, Medial lemniscus; LDTg, Laterodorsal tegmental nucleus; MnR, Median raphe nucleus; MPA, Medial preoptic area; MPO, Medial preoptic nucleus; MPOC, Medial preoptic nucleus, central part; MRe, Mammillary recess of the 3rd ventricle; ox, Optic chiasm; opt, Optic tract; OPT, Olivary pretectal nucleus; PaAP, Paraventricular hypothalamic nucleus, anterior parvicellular part; PaMP, Paraventricular hypothalamic nucleus, medial parvicellular part; PaPo, Paraventricular hypothalamic nucleus, posterior part; PaV, Paraventricular hypothalamic nucleus, ventral part; Pe, Periventricular hypothalamic nucleus; PMD, Premammillary nucleus, dorsal part; RMg, Raphe magnus nucleus; Py, Pyramidal tract; pr, Prepositus nucleus; scp, Superior cerebellar peduncle; Sch, Suprachiasmatic nucleus; SO, Supraoptic nucleus; sox, Supraoptic decussation; st, Stria terminalis; VLPAG, Ventrolateral periaqueductal gray; VMPO, Ventromedial preoptic nucleus; VMHA, Ventromedial hypothalamic nucleus, anterior part; VTA, Ventral tegmental area; VP, Ventral pallidum; 7, Accessory abducens/facial nucleus; 12, Hypoglossal nucleus. * Corresponding author. Tel.: +81 3 3784 8103; fax: +81 3 3784 6815. E-mail address: [email protected] (S. Shioda). 0143-4179/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2009.10.007
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(Lee et al., 1999). NPBWR1 and NPBWR2 mRNA has been detected at high levels in tissues of the human central nervous system (CNS) using RT-PCR analysis (Fujii et al., 2002). In particular, high levels of NPBWR1 mRNA were found in the hippocampus and amygdala (Brezillon et al., 2003) whereas, in the rat, strong NPBWR1 mRNA expression was detected in the hypothalamus and amygdala (Fujii et al., 2002). Similarly, in situ hybridization studies have revealed that NPBWR1 mRNA is present in the rat hypothalamus, including the arcuate nucleus (ARC), ventromedial nucleus (VMH), paraventricular nucleus (PVN), dorsomedial nucleus (DMH) and supraoptic nucleus (SON) (Lee et al., 1999), which are well known for their role in feeding regulation and energy homeostasis. Recently, several studies have reported the identification of two endogenous ligands for NPBWR1 and NPBWR2, neuropeptides W and B (NPW and NPB, respectively) (Fujii et al., 2002; Brezillon et al., 2003; Tanaka et al., 2003), which were isolated from the porcine hypothalamus. In the case of NPW, this has led to the characterization of two endogenous peptides consisting of 23- and 30-amino acid residues, termed neuropeptide W-23 (NPW23) and neuropeptide W30 (NPW30), respectively. Synthetic NPW23 and NPW30 have been shown to be similarly effective in binding and activating both NPBWR1 and NPBWR2 (Shimomura et al., 2002; Tanaka et al., 2003). In in vitro experiments, human NPW23 and NPW30 were shown to have a similar potency in terms of activating human NPBWR1. In rat, NPBWR1 is expressed in the hypothalamus, including its feeding centers, and administration of NPW to NPBWR1 knockout mice results in hyperphagic and decreased energy expenditure effects, suggesting a modulatory role for NPW in the control of feeding regulation. Recently, a physiological study reported that the intracerebroventricular (icv) injection of NPW induced acute food intake for the first 2 h in the light phase (Shimomura et al., 2002). However, Mondal et al. (Mondal et al. (2003)) reported that icv infusion of NPW significantly reduced dark-phase feeding 4 h after administration, an effect that was maintained for 48 h at higher doses, concomitant with increased body temperature and heat production. Administration of NPW30 has also been shown to increase arterial blood pressure heart rate and plasma catecholamine levels in rats (Yu et al., 2007), with Baker et al. demonstrating that administration of NPW23 elevates prolactin, corticosterone (Baker et al., 2003), and growth hormone levels. In addition, Niimi and Murao (Niimi and Murao (2005)) have reported that icv administration of NPW results in significant Fos expression in the PVN, suggesting that NPW is involved in stress-responsive signal transduction, and may be a modulator of the hypothalamus–pituitary–adrenal axis. On the other hand, the presence of NPBWR1 in the PVN is suggestive of a role in the modulation of neuroendocrine functions. Finally, Yamamoto et al. (Yamamoto et al. (2005)) have demonstrated that intrathecal administration of NPW23 or NPB in rats also suppresses inflammatory pain. Overall, these various studies reveal that NPW is a multifunctional peptide that mediates a range of physiological outcomes. Based on immunohistochemical analysis, NPW mRNA expression in the human CNS has been shown to be strongest in the substantia nigra, amygdala and hippocampus (Fujii et al., 2002; Singh et al., 2004). We (Takenoya et al., 2008), together with Dun et al. (Dun et al. (2003)) have also reported that NPW-like immunoreactive (NPW-LI) cell bodies are found in various regions of the rat brain, including the preoptic area (POA), PVN, SON, ARC, and dorsal and lateral hypothalamic areas, as well as the anterior and posterior pituitary gland. In contrast, Kitamura et al. (Kitamura et al. (2006)) reported expression in the Edinger–Westphal nucleus (EW), periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), and medial parabrachial nucleus (MPB), but saw no NPWLI cell bodies in the PVN. Given these somewhat conflicting results, the present study was designed to provide a detailed analysis of
NPW expression in the rat brain. This was achieved by first determining NPW gene expression by reverse-transcription polymerase chain reaction (RT-PCR), followed by immunohistochemical localization of NPW-LI neurons and the neuronal network between NPW-containing neurons and other hypothalamic neurons at the light and electron microscopic levels. 2. Materials and methods 2.1. Animals Male Wistar and Sprague–Dawley (SD) rats (Saitama Experimental Animal Supply, Saitama, Japan) weighing approximately 300 g were used. The animals were maintained on a 12/12 h light/dark cycle and supplied with standard laboratory chow and tap water ad libitum. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Showa University. Rats were placed under deep Nembutal anesthesia (40 mg/ kg, i.p.) and colchicine (200 lg/25 ll saline) injected into the third ventricle of the brain. After 48 h, the rats were perfused through the ascending aorta with 50 ml of saline (37 °C), followed by 250–300 ml of 2% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) fixative for 20 min. The brains were enucleated, trimmed, and immersed in the same fixative for 12 h at 48 °C. After washing, the fixed brains were transferred to a solution containing 20% sucrose in 0.1 M PB for 2 days at 4 °C. 2.2. RT-PCR The hypothalamic LH, ARC, VMH and PVN were punched out of the brain slices of the SD rats. The stomach was also taken from the SD rat. Total RNA was prepared from these tissues using TRIzol reagent (Invitrogen Corp), according to the manufacturer’s protocol. Total RNA from the stomach was used as a positive control for NPW (Mondal et al., 2003, 2006, 2008). Total RNA was treated with DNase (Roche, Basel, Switzerland). RNA was quantified spectrophotometrically and confirmed by ethidium bromide staining of 18S and 28S ribosomal RNA after electrophoresis on 1.0% agarose/formaldehyde gel. Four micrograms of total RNA were converted into cDNA using oligo d(T)12–18 primer (Invitrogen Corp.) and SuperScript III (Invitrogen Corp.) in a RT-reaction mixture (20 ll). PCR was performed using 0.8 ll of the RT-reaction mixture, 0.5 lM of each primer and AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster, CA, USA) in a total reaction volume of 25 ll. The primers used for rat NPW were 50 -GAGCTGTGGGAGGTA CGAAG-30 and 50 -CTGACAGGATCGGCAAAGAT-30 (GeneBank Accession No. AB084278). The PCR products were visualized by ethidium bromide staining under UV light following electrophoresis on a 1.5% agarose gel. After TA-cloning of PCR products, the nucleotide sequence of each inserted cDNA in the plasmid was confirmed by an automatic sequencing analyzer (ABI377, Applied Biosystems). 2.3. Preparation of sections Rats were anesthetized with pentobarbital (50 mg/kg, Dainippon Pharmaceutical, Saitama, Japan) then perfused with saline, followed by 250–300 ml of 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). The brain was transferred to 2% PFA/PB overnight at 4 °C, after which it was transferred first to 0.1 M PB solution containing 20% sucrose at 4 °C and then to 0.1 M PB solution containing 30% sucrose at 4 °C. The fixed brain was finally embedded in O.C.T compound in liquid nitrogen-cooled isopentane and stored at 80 °C. Sections 7 lm-thick were subsequently cut by cryostat (MICROM HM 500; MICROM, Heidelberg, Germany), and used for immunohistochemistry.
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2.4. Single staining
3.2. Localization of NPW-positive cell bodies and nerve fibers in brain
Sections were blocked in phosphate-buffered saline (PBS) containing 10% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA) for 60 min. Then, in order to detect NPW immunoreactivity, they were incubated overnight at 4 °C with anti-NPW antiserum (1:4000), (Mondal et al., 2003, 2006, 2008) washed three times with PBS for 5 min, and incubated with biotinylated anti-mouse IgG (1:400; DAKO, Carpinteria, CA, USA) for 2 h at room temperature (RT). The signals were amplified by ABC kit (Vector Laboratories, Inc.), and visualized using a peroxidase substrate (diaminobenzidine; DAB) kit (Vector Laboratories, Inc.) following washes with PBS. After dehydration, the mounted sections were coverslipped with malinol (Muto Pure Chemicals Ltd., Tokyo, Japan). NPW-like immunoreactivity (NPW-LI) was detected with the optical microscope (PROVIS, Olympus, Tokyo, Japan). As a control, the procedure described above was also performed omitting the primary antibody. Some frozen and vibratome sections were used as controls in which no anti-NPW antiserum or NPW-adsorbed NPW antiserum was used in the incubation process. No specific immunoreactivity was observed in these control sections.
NPW-LI cell bodies were observed in many regions of the rat brain (Figs. 2–4). NPW-positive cell bodies were seen in the islands of Calleja (ICj) (Fig. 2A and B), the ventrolateral hypothalamic nucleus (VLH) (Fig. 2D), the anterior parvocellular section of the paraventricular hypothalamic nucleus (PaAP) (Fig. 2E), the medial parvocellular section of the paraventricular hypothalamic nucleus (PaMP) (Fig. 2H), the posterior paraventricular hypothalamic nucleus (PaPe) (Fig. 2I), the lateral division of the posterior bed nucleus of the stria terminalis, (BSTLP) (Fig. 2F), the dorsal section of the dorsomedial hypothalamic nucleus (DMD) (Fig. 2K and L), the VMH (Fig. 3A and B), the ARC (Fig. 3C and D), LH (Fig. 3C and E), the dorsal premammillary nucleus (PDM) (Fig. 3F), the PAG (Fig. 3G), the EW (Fig. 3H), the central part of the lateral parabrachial nucleus (LPBC), the medial parabrachial nucleus (MPB) (Fig. 3I), the prepositus nucleus (Pr) (Fig. 3K), and the raphe nucleus (Fig. 3L). In addition, NPW-positive axon terminals were found in the lateral septal nucleus (LS) (Fig. 2C), the ventral part of the lateral division of the bed nucleus of the stria terminalis (BSTLV) (Fig. 2G), the amygdala (Fig. 2J), the LH (Fig. 2K and M), lateral parabrachial nucleus (LPB) (Fig. 3I) and the lateral PAG (LPAG) (Fig. 3J).
2.5. Electron microscopy
3.3. Electron microscopic observation
For electron microscopy, vibratome sections (40 lm thickness) that had been fixed as described above were first incubated in normal horse serum (1:20) for 20 min, and then in mouse anti-NPW antiserum (diluted to 1:1000) for 2 h at RT, followed by overnight incubation at 4 °C. On the following day, the sections were incubated with biotinylated anti-mouse IgG (DAKO, Carpinteria, CA, USA) for 1 h and then with ABC for 45 min at (Vectastain Elite ABC kit, Vector Laboratories). Following this, they were treated with DAB in 0.05 M Tris–HCl (pH 7.6) buffer including 0.005% hydrogen peroxide for about 3 min in the dark. After the DAB reaction, some of the sections were further treated with silver–gold intensification. These sections were post-fixed with 1% OsO4 in 0.1 M PB (pH 7.4) for 1 h at 4 °C, dehydrated in a graded ethanol series, and then embedded in a mixture of Epon–Araldite. Ultrathin sections were cut and examined by using a Hitachi H-7000 electron microscope. The specificity of the NPW antiserum used in this study has been reported elsewhere (Mondal et al., 2006).
At the electron microscopic level, many NPW-LI neurons were identified in the PVN (Fig. 5A). NPW-LI cell bodies often received inputs from other unknown neurons in the PVN (Fig. 5B), and NPW-LI was also demonstrated in dense-core vesicles (Fig. 5C). NPW-LI cell bodies often made synaptic contact with unknown axon terminals in the PVN (Fig. 5D). NPW-positive axon terminals were found to make synaptic contacts with unknown dendritic processes in the PVN (Fig. 5E), as well as the VMH (Fig. 5F). In the amygdala, NPW-like immunoreactivity was seen in dense-core vesicles (Fig 5G) and NPW-LI dendritic processes often received synaptic inputs from other unknown axon terminals (Fig. 5H).
3. Results 3.1. RT-PCR RT-PCR analysis was used to determine NPW mRNA expression in the rat hypothalamus. This revealed a PCR product of NPW corresponding to the predicted size of 211 bp (Fig. 1). NPW mRNA was expressed in the PVN, LH, ARC and VMH (Fig. 1). Expression levels of its mRNA in the PVN, LH, ARC and VMH were lower than that in the whole hypothalamus.
Fig. 1. RT-PCR analysis of NPW mRNA expression in the rat hypothalamic nuclei. RT-PCR analysis showed a PCR product corresponding to the predicted size of 211 bp.
4. Discussion In our present study, we observed distribution and localization of NPW-LI in rat brain but there is no clear species difference between Wistar and SD rats. There have been published two morphological study of NPW in brain by use of immunohistochemistry (Dun et al., 2003 and Kitamura et al., 2006). There are several different data on the distribution and localization of NPW-LI in brain tissues. Based on our present observation it may be caused from the different sources of the antibodies but not from the species difference. The present study is the first to demonstrate, based on RT-PCR analysis, that NPW mRNA is expressed in the PVN, LH, ARC, and VMH of the rat hypothalamus. In addition, we immunohistochemically examined the distribution of NPW-LI in rat brain pretreated with colchicine, a process which has recently been reported to dramatically enhance such immunoreactivity (Takenoya et al., 2008). Light microscopic observation revealed NPW-immunoreactive cell bodies in a number of areas, including the hypothalamus, midbrain, pons, and medulla oblongata. Many NPW-containing fibers were found in the LS, BST, amygdala, LH, PMD, PAG, and Pr. These results are schematically summarized in Fig. 4. We first identified at the electron microscopic level the presence of NPW-LI cell bodies in the PVN, VMH, and amygdala. These neurons were shown to make synaptic contact with unknown axon terminals in the PVN and ARC. A wide distribution of NPW-LI neurons in the brain suggests a role for NPW in feeding regulation as well as many physiological functions.
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Fig. 2. Photomicrographs of sections through colchicine-treated rat brain labeled with anti-NPW antiserum. NPW-positive cell bodies were seen in the islands of Calleja (ICj) (A and B), the ventrolateral hypothalamic nucleus (VLH) (D), the anterior parvocellular section of the paraventricular hypothalamic nucleus (PaAP) (E), the posterior part of the lateral division of the bed nucleus of the stria terminalis (BSTLP) (F), the medial parvocellular section of the paraventricular hypothalamic nucleus (PaMP) (H), the posterior paraventricular hypothalamic nucleus (PaPo) (I), the dorsal section of the dorsomedial hypothalamic nucleus (DMD) (K and L). NPW-positive axon terminals were seen in the lateral septal nucleus (LS) (C), the ventral part of the lateral division of the bed nucleus of the stria terminalis (BSTLV) (G), the amygdala (J), the LH (K and M). Scale bar = 250 lm (K), 100 lm (A, C, and F–I), 50 lm (B, D, E, J, L, and M). 3 V: 3rd ventricle.
Recently, it has been reported, based on in situ hybridization study, that NPW mRNA is expressed in the PAG, EW, and ventral tegmental area in the rat brain, but not in the PVN in mice and rats (Kitamura et al., 2006). However, in the present study, we identified NPW mRNA expression in the PVN as well as in the ARC, LH and VMH based on RT-PCR analysis. One plausible explanation for this discrepancy is that since the expression level of its mRNA is very low in the PVN, ARC, LH and VMH, it is difficult to detect NPW mRNA using in situ hybridization without signal amplification method such as tyramide signal amplification (TSA) system (Seki et al., 2008). NPBWR1 mRNA has previously been reported in the VMH (Williams et al., 2001), as well as the DMH, PVN, and ARC (Lee et al.,
1999). The abundance of NPBWR1 mRNA seen in the hypothalamus suggests that it may be involved in feeding regulation and metabolic disorders. In previous studies, icv administration of NPW suppresses food intake in the dark phase (Mondal et al., 2003). By contrast, during the light phase, NPW administration has been shown to stimulate feeding (Shimomura et al., 2002; Baker et al., 2003). We hypothesize that there are many NPW-mediated neural pathways from the ARC, VMH, and PVN to other brain areas, as a result of which NPW is able to act in concert with other neuropeptides to modulate the regulation of feeding behavior. A very few feeding-regulating peptides are isolated from the VMH (Sternson et al., 2005). Interestingly, in this study, we identified NPW-LI cell bodies in the VMH, a satiety center. Leptin acts on
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Fig. 3. Photomicrographs of sections through colchicine-treated rat brain labeled with anti-NPW antiserum. NPW-positive cell bodies were seen in the ventromedial hypothalamic nucleus (VMH) (A and B), the arcuate nucleus (ARC) (C and D), the lateral hypothalamus (LH) (C and E), the dorsal premammillary nucleus (PMD) (F), the periaqueductal gray (PAG) (G), the Edinger–Westphal nucleus (EW) (H), the central lateral parabrachial nucleus (LPBC) (I), the medial parabrachial nucleus (MPB) (I), the prepositus nucleus (Pr) (K), the raphe magnus nucleus (RMg) (L). NPW-positive axon terminals were seen in the lateral PAG (LPAG) (J), and the prepositus nucleus (Pr) (K). Scale bar = 250 lm (C), 100 lm (A, D, F, H, and J–L), 50 lm (B, E, G, and I). 3 V: 3rd ventricle. 4 V: 4th ventricle.
the VMH where its receptors are abundantly expressed, and reduces food intake. Changes of synaptic activity of projection from the VMH to the proopiomelanocortin (POMC) neurons in the ARC are known to inhibit feeding behavior (Elmquist et al., 1997; Koyama et al., 1998) and to regulate glucose level and energy homeostasis (Schwartz et al., 2000). Our present findings suggest that leptin acts on NPW-containing neurons in the VMH, and NPW may reduce food intake via POMC neuronal pathway. Further studies are required to identify neuronal interactions between NPW and POMC or leptin. The findings of the present study are similar to those reported by Dun et al. (Dun et al. (2003)), who reported NPW-LI cells in the PVH, supraoptic nucleus (SON), dorsal hypothalamus, LH, per-
ifornical nucleus, ARC, and posterior pituitary. It has also been reported that icv injection of NPW induced c-Fos expression in the rat hypothalamic areas such as the SON and PVN (Kawasaki et al., 2006). Injection of NPW at high dose into the LH increased food intake during 4 h after injection (Kawasaki et al., 2006). In contrast to the result obtained following NPW injection into the PVN, there was no effect during 24 h after injection (Levine et al., 2005). We have reported the first anatomical evidence of NPWcontaining efferent neuron population in the hypothalamus, and have shown that these NPW-containing neurons interact with orexin- and melanin-concentrating hormone-containing neurons in the LH (Takenoya et al., 2008). These data support the idea that feeding regulation is one of the important functions of NPW.
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Fig. 4. Schematic drawings showing the distribution of NPW-containing fibers (left) and cells (right) in the brain.
In the forebrain, we observed that the ICj receive inputs from the caudal part of the amygdala, which processes emotional memory (Ubeda-Banon et al., 2008) as well as from the septum, nucleus accumbens, and piriform cortex. In addition, the ICj also receives information from neurons in the substantia nigra and ventral tegmental area, located in the midbrain (Diaz et al., 1995).
We have found in this study that numerous NPW-LI cell bodies and fibers are observed in the POA and the BST which are involved in anxiety and the stress response. Similarly, the presence of NPBWR1 mRNA has been confirmed by in situ hybridization in the POA and BST (Tanaka et al., 2003). It is well known that NPW-containing neurons in the midbrain project to the central
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Fig. 5. Electron micrographs showing the NPW. (A) NPW-LI neurons were identified in the PVN. N, nucleus. (B) NPW-LI cell bodies are often received inputs from other unknown neurons (arrowhead) in the PVN. N, nucleus. (C) NPW-LI is demonstrated in dense-core vesicles as shown arrows. (D) NPW-LI cell bodies are makes synaptic contact (arrowhead) with unknown axon terminals in the PVN. (E) NPW-positive axon terminals (asterisk) are makes synaptic contact (arrowhead) with unknown dendrites processes in the PVN. (F) NPW-positive axon terminals (asterisk) are makes synaptic contact (arrowhead) with unknown dendrites processes in the VMH. (G) In the amygdara NPW-LI is observed in dense-core vesicles as shown arrows. (H) NPW-LI dendritic processes were often received (arrowhead) inputs from other unknown neurons in the amygdara. Scale bars = 0.2 lm.
amygdala (CeA) and BST. It is suggested that NPW is a regulator for the emotive responses, fear and anxiety by modulating the CeA and BST neurons (Kitamura et al., 2006; Shimomura et al., 2002).
The PAG is a region known to play a role in the descending modulation of pain and in defensive behavior (Monassi et al., 1999), with inputs from the spinomesencephalic tract in addition to affer-
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ent fibers from the hypothalamus, amygdala and brainstem. In addition, we found many NPW-LI cell bodies in the LPB, around the superior cerebellar peduncle, which are involved in the integration of taste information and the associated autonomic response (Matsuo et al., 1984). Fibers from the LPB are shown to project the amygdala and dorsal hypothalamus (Li et al., 2005; Saggu and Lundy, 2008), with the spinal cord–LPB–amygdala/hypothalamus pathway being known to participate in pain, anxiety and the autonomic nervous response (Day et al., 2004). Therefore, our present study provides the background on the involvement of NPW in modulation of response to anxiety, inflammatory pain (Kelly et al., 2005; Yamamoto et al., 2005; Singh and Davenport, 2006; Hondo et al., 2008). The LH, amygdala and BST are implicated to regulate feeding regulation and taste. In particular, nerve fibers in the LH are shown to project to premotoneurons in the NST and LPB (Matsuo et al., 1984). These premotoneurons are well known to innervate the salivary glands and tongue. NPW-containing cell bodies were found to be present in the LH, BST and LPB, in addition, NPW-containing fibers are present in the LPB. The above findings suggest that NPW is involved not only in regulation of food intake but also in saliva secretion, chewing and tongue movements. Further studies are required to analyze the precisely the neuronal network underpinning to understand each of these physiological functions. Acknowledgements This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture (F.T., H.K., S.S.), and a Hightechnology Research Center Project from the Ministry of Education, Science, Sports and Culture of Japan (S.S.). References Baker, J.R., Cardinal, K., Bober, C., Taylor, M.M., Samson, W.K., 2003. Neuropeptide W acts in brain to control prolactin, corticosterone, and growth hormone release. Endocrinology 144, 2816–2821. Brezillon, S., Lannoy, V., Franssen, J.D., Le Poul, E., Dupriez, V., Lucchetti, J., Detheux, M., Parmentier, M., 2003. Identification of natural ligands for the orphan G protein-coupled receptors GPR7 and GPR8. J. Biol. Chem. 278, 776–783. Day, H.E., Masini, C.V., Campeau, S., 2004. The pattern of brain c-Fos mRNA induced by a component of fox odor, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, suggests both systemic and processive stress characteristics. Brain Res. 1025, 139–151. Diaz, J., Levesque, D., Lammers, C.H., Griffon, N., Martres, M.P., Schwartz, J.C., Sokoloff, P., 1995. Phenotypical characterization of neurons expressing the dopamine D3 receptor in the rat brain. Neuroscience 65, 731–745. Dun, S.L., Brailoiu, G.C., Yang, J., Chang, J.K., Dun, N.J., 2003. Neuropeptide Wimmunoreactivity in the hypothalamus and pituitary of the rat. Neurosci. Lett. 349, 71–74. Elmquist, J.K., Ahima, R.S., Maratos-Flier, E., Flier, J.S., Saper, C.B., 1997. Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138, 839–842. Fujii, R., Yoshida, H., Fukusumi, S., Habata, Y., Hosoya, M., Kawamata, Y., Yano, T., Hinuma, S., Kitada, C., Asami, T., Mori, M., Fujisawa, Y., Fujino, M., 2002. Identification of a neuropeptide modified with bromine as an endogenous ligand for GPR7. J. Biol. Chem. 277, 34010–34016. Hondo, M., Ishii, M., Sakurai, T., 2008. The NPB/NPW neuropeptide system and its role in regulating energy homeostasis, pain, and emotion. Results Probl. Cell Differ. 46, 239–256. Kawasaki, M., Onaka, T., Nakazato, M., Saito, J., Mera, T., Hashimoto, H., Fujihara, H., Okimoto, N., Ohnishi, H., Nakamura, T., Ueta, Y., 2006. Centrally administered neuropeptide W-30 activates magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei with neurosecretion in rats. J. Endocrinol. 190, 213–223. Kelly, M.A., Beuckmann, C.T., Williams, S.C., Sinton, C.M., Motoike, T., Richardson, J.A., Hammer, R.E., Garry, M.G., Yanagisawa, M., 2005. Neuropeptide B-deficient mice demonstrate hyperalgesia in response to inflammatory pain. Proc Natl Acad Sci USA 102, 9942–9947. Kitamura, Y., Tanaka, H., Motoike, T., Ishii, M., Williams, S.C., Yanagisawa, M., Sakurai, T., 2006. Distribution of neuropeptide W immunoreactivity and mRNA in adult rat brain. Brain Res. 1093, 123–134.
Koyama, K., Shimabukuro, M., Chen, G., Wang, M.Y., Lee, Y., Kalra, P.S., Dube, M.G., Kalra, S.P., Newgard, C.B., Unger, R.H., 1998. Resistance to adenovirally induced hyperleptinemia in rats. Comparison of ventromedial hypothalamic lesions and mutated leptin receptors. J. Clin. Invest. 102, 728–733. Lee, D.K., Nguyen, T., Porter, C.A., Cheng, R., George, S.R., O’Dowd, B.F., 1999. Two related G protein-coupled receptors: the distribution of GPR7 in rat brain and the absence of GPR8 in rodents. Brain Res. Mol. Brain Res. 71, 96–103. Levine, A.S., Winsky-Sommerer, R., Huitron-Resendiz, S., Grace, M.K., de Lecea, L., 2005. Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake. Am. J. Physiol. Regul. Int. Comp. Physiol. 288, R1727–1732. Li, C.S., Cho, Y.K., Smith, D.V., 2005. Modulation of parabrachial taste neurons by electrical and chemical stimulation of the lateral hypothalamus and amygdala. J. Neurophysiol. 93, 1183–1196. Matsuo, R., Shimizu, N., Kusano, K., 1984. Lateral hypothalamic modulation of oral sensory afferent activity in nucleus tractus solitarius neurons of rats. J. Neurosci. 4, 1201–1207. Monassi, C.R., Leite-Panissi, C.R., Menescal-de-Oliveira, L., 1999. Ventrolateral periaqueductal gray matter and the control of tonic immobility. Brain Res. Bull. 50, 201–208. Mondal, M.S., Yamaguchi, H., Date, Y., Shimbara, T., Toshinai, K., Shimomura, Y., Mori, M., Nakazato, M., 2003. A role for neuropeptide W in the regulation of feeding behavior. Endocrinology 144, 4729–4733 (Epub 2003 Aug, 4727). Mondal, M.S., Yamaguchi, H., Date, Y., Toshinai, K., Kawagoe, T., Tsuruta, T., Kageyama, H., Kawamura, Y., Shioda, S., Shimomura, Y., Mori, M., Nakazato, M., 2006. Neuropeptide W is present in antral G cells of rat, mouse, and human stomach. J. Endocrinol. 188, 49–57. Mondal, M.S., Yamaguchi, H., Date, Y., Tsuruta, T., Shimbara, T., Toshinai, K., Shimomura, Y., Mori, M., Nakazato, M., 2008. Ontogeny of a new enteric peptide, neuropeptide W (NPW), in the developing rat stomach. Regul. Pept. 145, 141–146. Niimi, M., Murao, K., 2005. Neuropeptide W as a stress mediator in the hypothalamus. Endocrine 27, 51–54. O’Dowd, B.F., Scheideler, M.A., Nguyen, T., Cheng, R., Rasmussen, J.S., Marchese, A., Zastawny, R., Heng, H.H., Tsui, L.C., Shi, X., et al., 1995. The cloning and chromosomal mapping of two novel human opioid–somatostatin-like receptor genes, GPR7 and GPR8, expressed in discrete areas of the brain. Genomics 28, 84–91. Saggu, S., Lundy, R.F., 2008. Forebrain neurons that project to the gustatory parabrachial nucleus in rat lack glutamic acid decarboxylase. Am. J. Physiol. Regul. Int. Comp. Physiol. 294, R52–57. Schwartz, M.W., Woods, S.C., Porte Jr., D., Seeley, R.J., Baskin, D.G., 2000. Central nervous system control of food intake. Nature 404, 661–671. Seki, M., Kageyama, H., Takenoya, F., Hirayama, M., Kintaka, Y., Inoue, S., Matsuno, R., Itabashi, K., Date, Y., Nakazato, M., Shioda, S., 2008. Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla. Regul. Pept. 145, 147–152. Shimomura, Y., Harada, M., Goto, M., Sugo, T., Matsumoto, Y., Abe, M., Watanabe, T., Asami, T., Kitada, C., Mori, M., Onda, H., Fujino, M., 2002. Identification of neuropeptide W as the endogenous ligand for orphan G-protein-coupled receptors GPR7 and GPR8. J. Biol. Chem. 277, 35826–35832. Singh, G., Davenport, A.P., 2006. Neuropeptide B and W: neurotransmitters in an emerging G-protein-coupled receptor system. Br. J. Pharmacol. 148, 1033– 1041. Singh, G., Maguire, J.J., Kuc, R.E., Fidock, M., Davenport, A.P., 2004. Identification and cellular localisation of NPW1 (GPR7) receptors for the novel neuropeptide W-23 by [125I]-NPW radioligand binding and immunocytochemistry. Brain Res. 1017, 222–226. Sternson, S.M., Shepherd, G.M., Friedman, J.M., 2005. Topographic mapping of VMH ? arcuate nucleus microcircuits and their reorganization by fasting. Nat. Neurosci. 8, 1356–1363. Takenoya, F., Kitamura, S., Kageyama, H., Nonaka, N., Seki, M., Itabashi, K., Date, Y., Nakazato, M., Shioda, S., 2008. Neuronal interactions between neuropeptide Wand orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus. Regul. Pept. 145, 159–164. Tanaka, H., Yoshida, T., Miyamoto, N., Motoike, T., Kurosu, H., Shibata, K., Yamanaka, A., Williams, S.C., Richardson, J.A., Tsujino, N., Garry, M.G., Lerner, M.R., King, D.S., O’Dowd, B.F., Sakurai, T., Yanagisawa, M., 2003. Characterization of a family of endogenous neuropeptide ligands for the G protein-coupled receptors GPR7 and GPR8. Proc. Natl. Acad. Sci. USA 100, 6251–6256. Ubeda-Banon, I., Novejarque, A., Mohedano-Moriano, A., Pro-Sistiaga, P., Insausti, R., Martinez-Garcia, F., Lanuza, E., Martinez-Marcos, A., 2008. Vomeronasal inputs to the rodent ventral striatum. Brain Res. Bull. 75, 467–473. Williams, G., Bing, C., Cai, X.J., Harrold, J.A., King, P.J., Liu, X.H., 2001. The hypothalamus and the control of energy homeostasis: different circuits, different purposes. Physiol. Behav. 74, 683–701. Yamamoto, T., Saito, O., Shono, K., Tanabe, S., 2005. Anti-hyperalgesic effects of intrathecally administered neuropeptide W-23, and neuropeptide B, in tests of inflammatory pain in rats. Brain Res. 1045, 97–106. Yu, N., Chu, C., Kunitake, T., Kato, K., Nakazato, M., Kannan, H., 2007. Cardiovascular actions of central neuropeptide W in conscious rats. Regul. Pept. 138, 82–86.