神経内分泌 Neuroendocrine System
P1-2-131 ラット脳内におけるケトン体利用酵素の分布解析 A detailed mapping of the ketone body utilizing enzyme: Succinyl-CoA: 3-Ketoacid CoA-Transferase in rat brain ○森浩子1, 松田賢一2, 山脇正永1, 河田光博2 ○Hiroko Mori1, Ken-Ichi Matsuda2, Masanaga Yamawaki1, Mitsuhiro Kawata2 京都府立医大院　総合医療・医学教育学1, 京都府立医大院　生体構造科学2 Dept Medical Education, Kyoto Prefectural Univ, Kyoto, Japan1, Dept Anatomy and Neurobiology, Kyoto Prefectural Univ, Kyoto, Japan2 Ketone bodies are the energy-rich compounds produced in the liver as a result of fatty acid metabolism. β-hydroxybutyrate, the principal ketone body displaces glucose as the major source for brain when glucose is not readily available. To reveale detailed distribution pattern of the enzyme for ketone body catabolism: succinyl-CoA: 3-ketoacid CoA-transferase (SCOT), we carried out immunohistochemical study and detailed quantification or rating of SCOT immunoreactive (-ir) neurons in the rat brain. Density was expressed as the percentage of ir positive cells related to total cell number defied by Nissl staining. In the cerebral cortex, the strong SCOT-ir were observed in cingulate cortex (85%), agranular nucleus (83%), perirhinal cortex (80%), piriform cortex (61%) and the pyramidal cell layer in neocortex (62-90%). In the hippocampal formation, SCOT-ir neurons predominantly exist in stratum pyramidale of CA1, CA2 and CA3 (80-85%) and granular layer of dentate gyrus (52%), while no immuno-positive neuron was observed in stratum radiatum and stratum lacunosum-moleculare. SCOT-ir neurons were also distributed in the amygdala (34-62%), medial preoptic nucleus (24%), anteroventral periventricular nucleus (36%), ventromedial nucleus of the hypothalamus (52%) and thalamic nuclei with low to moderate immunoreactivities. However, there were no SCOT-ir were observed in basal ganglia, habenular nucleus, suprachiasmitic nucleus, anterior hypothalamic area and mammillary nuclei and its surrounding structures. These observations revealed regional difference in SCOT expression in the brain, providing an insight into brain energetics in termes of ketone body utilization.	P1-2-132 ラット海馬シナプスにおけるステロイドの合成とその作用 Hippocampus-synthesized steroids and their effect on synaptic plasticity in the rat hippocampal synapses ○北條泰嗣1,2, 向井秀夫1,3, 山崎岳4, 木本哲也1,2, 川戸佳1,2 ○Yasushi Hojo1,2, Hideo Mukai1,3, Takeshi Yamazaki4, Tetsuya Kimoto1,2, Suguru Kawato1,2 東京大院　総合文化1, JST Bioinformatics Project2, 埼玉医大・生化学3, 広島大・総合科学4 Grad. Sch of Arts and Sci., Univ. of Tokyo, Tokyo1, JST BIRD2, Saitama Med. Univ.3, Hiroshima Univ.4 Hippocampal synaptic plasticity is modulated by steroid hormones which had been believed to be released from testis, ovary or adrenal gland, and reach hippocampal synapses via blood circulation. However, we revealed (1) hippocampus itself synthesizes steroids and (2) these hippocampus-synthesized steroids modulate synaptic plasticity. Steroidogenic enzymes were mainly localized in hippocampal neurons, especially, in synapses. Hippocampus synthesized estrogen (estradiol: E2), androgens (testosterone: T, dihydrotestosterone: DHT), and corticosteroids. Concentrations of E2, T and DHT in the hippocampus were much higher than those in plasma (Higo et al., 2011 PLoS ONE; Hojo et al., 2009 Endocrinology; Hojo et al., 2004 PNAS). These results suggest that synaptic plasticity is modulated by hippocampus-synthesized steroids rather than circulation-derived steroids. E2, T and DHT increased total spine density of CA1 pyramidal neurons. E2 mainly increased small-head spines (<0.4 μm) whereas DHT mainly increased large-head spines (>0.5 μm) with large efficacy of neurotransmission. We discriminated the effect between estrogen and androgen on spinogenesis for the first time. We developed new soft ware, Spiso-3D, for the automated detection of spines based on analysis of geometrical features (JST BIRD, Mukai et al., 2011 Cerebral Cortex). Spiso-3D reconstructs dendrite, detects spines and outputs the distribution of spine head diameter using both eigenvalue images and information of brightness processed from original image files. Spiso-3D enabled us to distinguish the effect of androgen and estrogen on spinogenesis, which leads to advance our understanding of function of hippocampus-synthesized steroids.
P1-2-133 音楽聴取がステロイドホルモンに及ぼす影響について-受容体多型と音楽能力との関係 Influence of the music listening on Steroid Hormone, and a relation with the receptor polymorphism and the musical ability ○福井一1, 豊島久美子1 ○Hajime Fukui1, Kumiko Toyoshima1 奈良教育大学教育学部1 Dept Edu, Nara Univ of Edu, Nara1 In this study, the influence that music listening has upon steroid hormone values, and the relationship of steroid hormone receptor polymorphism and musical ability was examined. Steroid hormone receptor is chosen as a center of the study with several reasons; many of the biological actions of steroid hormones are mediated by intracellular receptors, which exert transcriptional control of steroids-dependent genes and are expressed in various regions of the brain. In addition, cell membrane receptors are present for several steroid hormones, where cellular responses are much quicker than the intracellular receptors. Lastly, together with music listening, receptor polymorphism is thought to affect on hormone changes and musical ability. 30 adolescents, 15 males and 15 females, were chosen as subjects in this experiment. They were given 1. Chill-induced music, 2. Disliked (Detestable) music and 3. Silence (controlled) as music stimuli for 5 minutes each. Before and after the experiments, saliva was taken to measure hormones - testosterone, estradiol and cortisol. Simultaneously, the subjects' emotional state was examined using STAI and SACL before and after the stimuli. Saliva was taken separately to examine whether the subjects were to have polymorphism of androgen receptor or estrogen receptor. Gordon Musical Talent Test was employed to determine the subjects' musical ability. Additionally, in order to determine the relationship to their personality, NEO-PT-R was conducted and the subjects' music experiences were examined too. As a result, when listened to chill-induced music, cortisol values were significantly lowered in both sex, but no conspicuous changes were seen when listened to disliked music or silence. The value of testosterone decreased with male, increased with female with chill-induced music. No remarkable changes were measured with disliked music or silence. No striking changes considering estradiol values seen under any musical stimuli.	P1-2-134 ヒト副甲状腺ホルモン製剤テリパラチドの皮下投与の中枢神経系に及ぼす影響 The effect of subcutaneous injection with teriparatide, human parathyroid hormone (1-34), on the central nervous system in adult male rats ○杉本皓司1, 山下賢也1, 加藤直人2, 首藤文洋1, 久野節二1 ○Koji Sugimoto1, Kenya Yamashita1, Naoto Kato2, Fumihiro Shutoh1, Setsuji Hisano1 筑波大院・人間総合科学・感性認知脳科学1, 旭化成ファーマ2 1. Lab. Systems Brain Sciences, Kansei, Behavioral and Brain Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki1, Asahikasei Pharma Corporation, Tokyo2 Teriparatide, human parathyroid hormone (1-34), which is a therapeutic agent for osteoporosis, has an effect to increase the bone mineral density and to prevent bone fractures in postmenopausal women with osteoporosis. Unfortunately, however, it has been reported that teriparatide might have undesirable side effects such as nausea and vomiting. The mechanism of nausea is still unclear, despite it has been suggested that this syndrome is often accompanied by vasopressin (VP) release and subsequent vasoconstriction. The aims of this study are to investigate an effect of subcutaneously injected teriparatide on the central nervous system, particularly neuroendocrine neurons in the paraventricular (PVN) and supraoptic nuclei (SON) of rats. Adult male Sprague-Dawley strain rats were subjected to subcutaneous injection with saline (1 ml/kg/day) for consecutive 7 days before experiment. On experiment day, teriparatide acetate dissolved in saline (at doses of 0 (saline alone), 13.6, 27.2, and 40.8 µg/kg) was administered subcutaneously to the everyday saline-injected rats, and perfusion-fixed 120 min later. Brains containing the PVN and SON were frozen, and cut into coronal sections. The sections were processed for immunostaining of c-Fos, which is an excited neuron marker, and for in situ hybridization for VP mRNA. Teriparatide-induced c-Fos expression occurred in the ventral part (the VP neuron-rich region) of the SON and the ventral medial parvocellular subdivision (mpv) of the PVN. These data suggest that teriparatide has a stimulatory effect on the neuronal activity of the magnocellular VP neurons in the SON and autonomic-related mpv neurons, indicating some central effect of peripherally injected hormonal analogs.
P1-2-135 持続的ストレス負荷により下垂体のメラノトロフに発現するVGFとその発現制御 Continuous stress induces expression of VGF in melanotrophs of pituitary gland ○時實恭平1, 小西博之1, 安井正佐也1, 小川登紀子3, 佐々木一樹2, 南野直人2, 木山博資1 ○Kyouhei Tokizane1, Hiroyuki Konishi1, Masaya Yasui1, Tokiko Ogawa3, Kazuki Sasaki2, Naoto Minamino2, Hiroshi Kiyama1 名古屋大院・医・機能組織1, 国立循環研セ・分子薬理2, 大阪市大院・医・システム神経3 Dept. Functional Anatomy and Neuroscience, Nagoya Univ, Grad. Sch. Med., Nagoya, Aichi1, Dept. Molecular Pharmacology, National Cerebral and Cardiovascular Center Research Institute, Osaka2, Dept. Physiology, Osaka City Univ. Grad. Sch. Med., Osaka3 Prolonged exposure to physiological and mental stresses elicits profound effects on homeostasis, which may lead to cryptogenic disorders such as chronic fatigue syndrome (CFS). Although currently there are no established animal models that provide pathophysiological insights into the syndrome, the rat continuous stress (CS) model partially mimics the symptoms associated with the syndrome. In this model, a rat is placed in a cage filled with a very low level of water (1.5 cm depth) for few days, to apply continuous multiple stresses. Using this model we have revealed that the pituitary represents one of the most affected organs. Here we show that mRNA for VGF (non-acronymic), a member of the granin family, was induced specifically in the intermediate lobe (IL) under CS. The VGF gene encodes a 617 amino acid protein in rodents that undergoes processing to yield a number of biologically active peptides. Immunohistochemistry with antibody against the C-terminal end of VGF peptide (VGF-C) revealed the increase in VGF expression in a subset of melanotrophs. However, antibodies against neuroendorine regulatory peptides (NERP1-3), each of which specifically recognizes distinct internal sequences in VGF, did not show increased immunoreactivity in pituitary, suggesting that CS induces VGF protein expression but does not promote precursor processing to generate processing products such as NERPs. An in vitro study using primary cultured melanotrophs showed a direct effect on VGF inductions by dopamine suppression. Moreover, administration of the dopamine D2 receptor agonist bromocriptine prevented the inductions of VGF in the IL of CS rats, whereas application of the dopamine D2 antagonist sulpiride induced significant expressions of VGF in IL of normal rats. In CS animal dopaminergic innervation from hypothalamus was suppressed in IL. These results suggested that the VGF expression in the IL was negatively regulated by dopamine originated from hypothalamus.	P1-2-136 脳海馬で合成される性ステロイドによる神経シナプス可塑性の急性制御 Acute Modulation of Synaptic Plasticity of Pyramidal Neurons by Hippocampal-derived Sex Steroids ○長谷川賢卓1,2, 堀田佳佑1, 向井秀夫1大石悠貴1, 北條泰嗣1,2, 川戸佳1,2 ○Yoshitaka Hasegawa1,2, Keisuke Hotta1, Hideo Mukai1, Bon-chu Chung2,3, Yuuki Ooishi1, Yasushi Hojo1,2, Suguru Kawato1,2 東京大院・総合文化1, 国際共同研究　（科学技術振興機構）2, 中央研究院　生命科学部　分子生物研究所3 Dept Biophysics and Life Sciences, Univ of Tokyo, Tokyo1, International collaboration (JST)2, Institute of Molecular Biology, Taiwan3 Abstract: Hippocampal-derived sex hormones are neuromodulators in the hippocampus. We investigated effects of Estradiol(E2), Testosterone(T) and Dihydrotestosterone(DHT). Adult hippocampus synthesized 8nM E2. Synaptic estrogen receptors(ERα) are in spines of hippocampal neurons. Localization of ERαin spines was demonstrated by immunogold electron microscopy. We demonstrated that E2 induced the long term potentiation (LTP) of the hippocampal neurons by weak theta burst stimulation. E2 also increased the density of spines in CA1 pyramidal neurons in hippocampal slices. Effects of E2 are induced by mitogen-activated protein kinase (MAPK) in hippocampal neurons.
P1-2-137 神経内分泌における血管系ダイナミックスの意義 Dynamic reorganization of vascular structure is crucial in hypothalamo-neurohypophysial neurosecretion ○宮田清司1, 森田晶子1,2 ○Seiji Miyata1, Shoko Morita1,2 京都工芸繊維大院・応用生物1, 奈良県立医大・医・第二解剖2 Dept of Appl Biol, Kyoto Inst of Technol, Kyoto, Japan1, Dept of Aant & Neurosci, Nara Med Univ, Nara, Japan2 The concept of neurosecretion, the production and secretion of neuropeptides by brain neurons and the actions of these hormones at target tissues in mammalians, is proposed by Scharrer and Scharrer in 1928. Later, Bargmann has identified that hypothalamic neurons secrete arginine vasopressin (AVP) and oxytocin (OXT) from their axonal terminals of the neurohypophysis (NH) into the blood circulation. Neurosecretion is the release of peptides into the circulation from hypothalamic neuronal terminals for controlling growth, maturation, reproduction and fluid homeostasis, however, dynamic reconstruction of fenestrated vasculature in neurosecretion is unknown. Here, we demonstrated continuous proliferation of endothelial cells by the signaling of astrocyte-derived vascular endothelial cell growth factor-A in the neurohypophysis of normal adult mice. Neurohypophysial perivascular protrusions, consisting of pericyte cellular processes and extracellular matrix, function as a main passing route of low-molecular-mass molecules and a chronic salt-loading stimulation to promote vasopressin secretion, elevated vascular permeability of low-molecular-mass molecules concomitant with increased number of perivascular protrusions. Secretion-dependent reconstruction of perivascular structure was due primarily to pericyte shape conversion by neuronal-terminal-derived platelet-derived growth factor-BB. The present study, thus, demonstrates participation of dynamic vascular reconstruction in hypothalamo-neurohypophysial neurosecretion, indicating that neurohypophysial vasculature has more dynamic nature than previously thought and shed light on a new aspect for understanding neurosecretion.	P1-2-138 エストロゲン受容体α陽性神経細胞のフィードバック応答の可視化 Visualization of feedback responses of estrogen receptor α-positive neurons ○松田賢一1, 柳沢美歩2, 佐野一広3大越幸太2, 塚原伸冶2, 小川園子3, 河田光博1 ○Ken Ichi Matsuda1, Miho Yanagisawa2, Kazuhiro Sano3, Sergei Musatov4, Kota Okoshi2, Shinji Tsukahara2, Sonoko Ogawa3, Mitsuhiro Kawata1 京都府立医大院・医・生体構造1, 埼玉大院・理工・生命科学2, 筑波大院・人間総合科学・行動神経内分泌3 Dept Anat Neurobiol, Kyoto Pref Univ Med, Kyoto1, Life Sci, Grad Sch Sci Engin, Saitama Univ, Saitama2, Behav Neoroendocrinol, Grad Sch Comprehen Hu Sci, Univ Tsukuba3, Neorologix Inc, Fort Lee, USA4 Estrogen receptor (ER) α plays important roles in the development and function of various neuronal systems through activation by its ligand, estrogens. To visualize ERα-positive neurons, we generated transgenic (tg) mice expressing green fluorescent protein (GFP) under the control of the ERα promoter. GFP-positive neurons were observed in areas previously reported to express ERα mRNA, including the bed nucleus of the stria terminalis (BNST), medial preoptic area (MPO). In these areas, GFP signals mostly overlapped with ERα immunoreactivity. We examined the effect of estrogen deprivation by ovariectomy (OVX) on the structure of the GFP-positive neurons. The area of ERα-positive cell bodies in the BNST and MPO was measured by capturing the GFP signal and was found to be significantly smaller in OVX mice than in control mice. When neurons in MPO were infected with an adeno-associated virus which expressed shRNA targeting ERα gene, an apparent induction of GFP was observed in the MPO, suggesting a negative feedback mechanism in which ERα controls expression of the ERα gene itself. Thus, the ERα promoter-GFP tg mice will be useful to analyze estrogen signaling mechanism in the brain.
P1-2-139 海馬スパインにおけるエストロゲンとアンドロゲンの効果 Effects of estrogen and androgen on the hippocampal spines ○吉屋美雪1, 川戸佳1 ○Miyuki Yoshiya1, Suguru Kawato1 東京大学大学院 総合文化研究科 広域科学専攻1 Grad Sch of Arts and Sci, Univ of Tokyo, Tokyo, Japan1 Dendrotic spines are postsynaptic structures and they store the memory in the hippocampus. We already demonstrated that sex-steroids are synthesized not only in the gonads but also locally in the hippocampus (Hojo et al., 2004, 2009). Here, we focus on the rapid effect of testosterone (T) dihydrotestosterone (DHT) and estradiol (E2), on the synaptic plasticity via synaptic steroid receptors. We examined the rapid modulation of spines by application of sex-steroids, by means of single spine analysis of Lucifer-Yellow injected neurons in adult male hippocampal slices. 3D images were obtained using confocal microscope. Following a 2 h treatment with T, DHT and E2 in CA1 region, the density of spines was significantly increased by approximately 1.3 fold of that in control (approx 1.00 spines/um). Mathematic automated analysis (by Spiso-3D software) of spine morphology distinguished different effects of these three sex-steroids (Mukai et al., 2011). DHT increased large-head and middle-head spines, T increased large-head and small-head spines, but E2 increased small-head spines. To determine the pathway of sex-steroid signaling, we applied various kinase inhibitors including the inhibitors of MAP kinase, PKA, PKC and PI3K, etc. Many of these inhibitors suppressed the increase of spines. These results indicate that androgen and estrogen rapidly drive the multiple kinase pathways resulting in actin polymerization. Synaptic AR and ER receptors are also included. These results show molecular mechanisms of sex-steroid-induced neurotrophic effects on synapses.	P1-2-140 ES細胞由来AVP産生ニューロン培養系におけるRab3a、Rabphilin3aのAVP分泌への関与の検討 Involvement of Rab3a and Rabphilin3a in AVP secretion in ES cell-derived AVP neurons ○清田篤志1, 椙村益久1, 竹内誠治1, 泉田久和1, 落合啓史1, 藤沢治樹1, 高木博史1, 福岡一貴1, 須賀英隆1, 渡辺崇2, 長崎弘3, 大磯ユタカ1 ○Atsushi Kiyota1, Yoshihisa Sugimura1, Seiji Takeuchi1, Hisakazu Izumida1, Hiroshi Ochiai1, Haruki Fujisawa1, Hiroshi Takagi1, Kazuki Fukuoka1, Hidetaka Suga1, Takashi Watanabe2, Hiroshi Nagasaki3, Yutaka Oiso11 名古屋大学大学院医学系研究科　糖尿病・内分泌内科学1, 名古屋大学大学院医学系研究科　神経情報薬理学2, 藤田保健衛生大学　生理学I3 Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine1, Department of Cell Pharmacology, Nagoya University Graduate School of Medicine2, Department of Physiology, Fujita Health University3 Small GTPase Rabs, Rab effectors, and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated to play a central role in regulated exocytosis of secretary vesicles. The molecular mechanism underlying the involvement of these proteins in the regulation of arginine vasopressin (AVP) secretion remain to be elucidated. Recently, it has been reported that embryonic stem (ES) cells differentiate into rostral hypothalamic progenitor cells including AVP neurons when dissociated ES cells are quickly reaggregated in low cell-adhesion culture wells and cultured as floating aggregates in growth factor free chemically defined medium (SFEBq/gfCDM culture). The aim of this study is to examine the involvement of Rab proteins in AVP secretion using SFEBq/gfCDM cultured ES-cell aggregates (SgESa). The expression of Rab3a, its effector, rabphilin3a, and SNAP25, a t-SNARE protein in the SgESa was detected by immunoblotting. Incubating SgESa in high K+ and hyperosmotic media caused a significant increase in AVP secretion. Immunocytochemical analysis of ES cell-derived AVP neuron (ES-AVP cells) showed that rabphilin3a was distributed in the subplasma membrane region, and partially colocalized with copeptin, a surrogate marker of AVP. After the high K+ stimulation, the distribution of rabphilin3a seemed to be enriched in plasma membrane region with copeptin. Co-immunoprecipitation and pull-down assay revealed an association of Rabphilin3a with Rab3a and SNAP25 with AVP. Rph3a knockdown by siRNA in dispersed SgESa resulted in a significant inhibition of the high K+ stimulated AVP secretion. Finally, overexpression of EGFP-tagged AVP fused protein (AVP-EGFP) into ES-AVP cells enabled the time-lapse imaging of AVP vesicles. AVP-EGFP could be a useful tool to investigate processes of AVP secretion.
P1-2-141 飼育温度依存的な低温耐性は、神経ホルモンであるインスリンが腸と神経で受容されることで成立する Cultivation temperature-dependent cold tolerance is regulated by a neuronal hormone insulin that is received by intestine and neuron in C. elegans ○太田茜1, 宇治澤知代2, 井上琢朗1, 桑原直人1, 井上奈穗1, 久原篤1 ○Akane Ohta1, Tomoyo Ujisawa2, Takurou Inoue1, Naoto Kuwahara1, Naho Inoue1, Atsushi Kuhara1 甲南大学　理工学部　生物学科1, 甲南大院　自然科学　生物2 Dept Biol, Konan Univ, Kobe1, Dept Biol, Grad Sch Nat Sci, Konan Univ, Kobe2 Temperature is one of the most critical environmental stimuli and cause biochemical changes in the body. Animals including human, therefore, can respond and adapt to the changes in ambient temperature. We are investigating about the molecular mechanism underlying a cultivation temperature-dependent cold tolerance in nematode C. elegans. 25°C-cultivated animals were died by cold stimuli 2°C. By contrast, most of 15°C-cultivated animals can survive at 2°C. In order to determine the molecular pathway for this cultivation temperature-dependent cold tolerance, we tested various mutants. We found that the mutants defective in DAF-2/insulin receptor or its downstream molecules showed abnormal cold tolerance. DAF-2 is an only insulin receptor in C. elegans, while there are about 40 ligands for insulin receptor. We have found that at least three insulin-like molecules, DAF-28, INS-6 and INS-1, are essential for cold tolerance. Two insulins, DAF-28 and INS-6, are both positive agonists and work redundantly for DAF-2/insulin receptor in cold tolerance. Genetic epistasis analysis indicated that INS-1/insulin genetically inhibits DAF-2/insulin receptor through a negative regulation of DAF-6/insulin. Unexpectedly, cell specific rescue experiments revealed that DAF-28/insulin is released by a single sensory neuron ASJ that is known as a light-sensing neuron, and that DAF-2/insulin receptor functions in both "intestine" and "neuron". These results suggest that a single sensory neuron regulates the cultivation temperature-dependent cold tolerance of animal through intestine and neuron. We are now investigating whether a light-sensing neuron ASJ acts as a temperature-sensing neuron by the calcium imaging analysis.	P1-2-142 視床下部領域に新たに同定されたペリニューロナルネット陽性細胞を含む神経核について A recently identified nucleus in the hypothalamus containing perineuronal nets-associating neurons ○堀井謹子1, 笹川誉世1, 松永渉1, 橋本隆1, 西真弓1 ○Noriko Horii1, Takayo Sasagawa1, Wataru Matsunaga1, Takashi Hashimoto1, Mayumi Nishi1 奈良県立医大　医・第一解剖1 Dept Anat Cell Biol, Nara Med Univ, Nara1 Perineuronal nets (PNNs) are known as a specialized form of extracellular matrix in the adult brain. PNNs are mainly composed of chondroitin sulfate proteoglycans accumulating around somata and dendrites of specific types of neurons. PNNs are thought to inhibit functional plasticity and stabilize neural networks in the adult brain and their formation is started at the end of the critical period. In the present study, we found a novel cluster of hypothalamic neurons possessing PNNs and clarified the cytological and anatomical characteristics of this cluster. WFA (a lectin for PNNs marker) labeling showed that intensely stained PNNs were observed in a restricted region neighboring the paraventricular nucleus (PVN), fornics and the central part of the anterior hypothalamic area (AHC) in the brain of adult C57BL/6 mice. Since this region has not been identified as a specific region, we performed double/single labeling analysis using WFA and Nissl to assess whether this region could be identified as a nucleus. WFA-labeled hypothalamic region was discriminable by single Nissl labeling and the density of Nissl in this region was lower than that in neighboring nuclei including the PVN and AHC. Such WFA-labeled hypothalamic region was observed in other strains of mice (ICR, BALB/c) and slightly in Wistar rat. Further histological analysis revealed that some PNNs in this region were formed around GABAergic but not parvalbumin-expressing neurons and interestingly some around enkephalinergic neurons. Anterograde tracing using biotinylated dextran amine (BDA) revealed that BDA-labeled fibers were observed in the lateral septal nucleus with colocalization with enkephalin. From these results, we propose that this WFA-labeled region in the hypothalamus neighboring the PVN and fornics is a new subdivision of the perifornical area predominantly composed of GABAergic and enkephalinergic neurons. We are now investigating functions of this region.

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