キュウキュウ ニオケル ステロイド ゴウセイ コウソ ノ キョクザイ

Neurosteroids are synthesized through mechanism at least partly independent of the peripheral steroidogenic glands, and their neurobiological actions seem to depend on the specific functions of various brain regions. However, little is known about neurosteroids function corresponding to specific structure and functions of the brain regions. Thus the present study analyzed localization of steroid-synthesizing enzymes in the rat and mouse olfactory bulb. RT-PCR and Western-blotting indicated the possible presence of almost all enzymes of steroid synthesis from cholesterol to estradiol, that is, cholesterol side chain cleavage enzyme (P450scc : cholesterol→pregnenolone), 3β-hydroxysteroid dehydrogenase/Δ5 - 4 isomerase (3β-HSD : pregnenolone→progesterone), 17α-hydroxylase/C17-C20lyase (P450c17 : progesterone→ androstenedione), 17β-hydroxysteroid dehydrogenase (17β-HSD : androstenedione→testosterone), 5α-reductase (5αR:testosterone→5α-dihydrotestosterone), and aromatase (testosterone→ estradiol). Immunohistochemistry confirmed that the 5α-reductase was mainly in glial cells with various immunoreactivity and co-localization pattern, but other enzymes were in bulbar neurons, and at least co-localized in mitral/tufted cells. In addition, so far analyzed, enzymatic activities of P450c17 (a key enzyme in sex steroid synthesis) and 17β-HSD were detected biochemically. These enzyme expression and activities were influenced under exposure of 17β-estradiol with various degrees. These findings indicated the presence of steroid-synthesizing activities, and thus of possible neurosteroid metabolism in the olfactory bulb

ラット嗅球神経回路における非GABA系介在ニューロンのシナプス結合の微細構造解析

嗅球は比較的少数のニューロン種から構成される明瞭な層構造を持つ脳の領域である.投射ニューロンである僧帽細胞/房飾細胞は嗅球の表層の糸球体で匂い情報を受け,細胞体に至る過程で種々の介在ニューロンからの調整を受け,処理された情報を高次中枢へ投射する.情報入力部である糸球体には傍糸球体細胞(JGニューロン)が多数分布しており,形態的および化学的性質からtype 1 JGニューロンとtype 2 JGニューロンの2種の介在ニューロン群に分類される.γ-アミノ酪酸免疫陽性ニューロン(GABAニューロン)とcalbindin 免疫陽性ニューロン(CBニューロン)およびcalretinin免疫陽性ニューロン(CR ニューロン)はそれぞれのtype 1 JGニューロンとtype 2 JGニューロンの代表的なニューロン群である.これらのニューロンは対称性シナプスを形成し,抑制性に働くと考えられているが,神経回路内でシナプス形成するニューロンが異なることから,匂い情報の処理過程において異なる働きをしていることが考えられる.また,ラット嗅球のtype 2 JGニューロンは抑制性の神経伝達物質であるGABAに免疫陰性を示すが,その伝達物質の詳細は不明である.そのため本研究では電子線トモグラフィーによりGABA免疫陽性ニューロンとGABA免疫陰性ニューロンが形成するシナプスの詳細な解析を行った.今回解析したシナプスでは,GABAニューロンとCBニューロンのシナプス間隙の大きさに有意差が認められ,GABAニューロンとCBおよびCRニューロン間でシナプス小胞の大きさに有意差がみられた.また,CBニューロンとCRニューロンの間においても小胞に形態的な違いがみられた.以上の結果から,同じGABA免疫陰性ニューロンでも含有する化学物質によってシナプスの形状が異なり,機能的特性が異なることが示唆された.The olfactory bulb (OB) is a brain region with distinct layers organized by projection neurons, mitral/tufted cells and different kinds of interneurons. Mitral/tufted cells extend their dendrites to glomeruli in the superficial part of the OB. Here they receive odor information and send it to higher brain regions through their axons. Mitral/tufted cells are regulated by juxta-glomerular (JG) neurons in the glomeruli. JG neurons, which surround the glomerulus, have been classified into two types, type 1 and type 2, based on morphological and chemical characteristics. Type 1 neurons are γ-amino butyric acid (GABA)-immunoreactive neurons, and type 2 neurons are calbindin (CB) and calretinin (CR)-immunoreactive neurons.Type 1 2 interneurons might play different roles in odor information processing due to their synaptic contacts onto different neuronal types. Although both types of interneurons form symmetrical synapses morphologically and because of this are expected to function as inhibitory, these synapses could have distinct functions. Interestingly, type 2 interneurons in the rat OB have been shown to be immunonegative for GABA, which is the major neurotransmitter of inhibitory synapses; their transmitter remains to be identified. Therefore, in the present study, we have conducted ultrastructural analysis of synapses formed by GABA-immunopositive and -immunonegative JG neurons by electron microscopic tomography. Results indicate statistical differences in the width of synaptic clefts between GABA- and CB-immunopositive neurons and in the size of synaptic vesicles between GABA-, CB-, and CR-immunopositive neurons. In addition, we have found some differences in the shape of synaptic vesicles between CB- and CR-immunopositive neurons. Together with the findings of our previous studies, we have shown distinct structural differences of synapses in subgroups of type 2 neurons and differences between type1 and 2 interneurons. These structural differences differ with the different proteins expressed in the interneurons and could indicate different functional properties of the subgroups of interneurons

シナプスマーカーVGLUT1, VGAT を用いた嗅球神経回路の新たな形態学的解析

嗅覚の一次中枢である嗅球の投射ニューロン（僧帽細胞，房飾細胞）は，その樹状突起上で樹状突起間シナプスを形成したのちに，高次の脳中枢に情報を伝達する．従って，僧帽細胞の突起上のシナプスの分布を明らかにすることは，匂い情報調節を解析する上で必須である．従来，シナプス結合を形態学的に同定するものは高解像度な電子顕微鏡による解析であった．しかし，電子顕微鏡では，観察できる領域は限られ，広範囲の解析を行うのは困難であり，僧帽細胞を厳密に同定することもできない．Vesicular glutamate transporter（VGLUT1）やvesicular GABA transporter（VGAT）などのシナプスマーカーの有用性が最近多くの脳領域で報告されており，これらのマーカーにより電子顕微鏡による解析以上に，嗅球でも信頼性の高いシナプスの定量ができるかもしれない．本研究では，新たなマーカーによる，樹状突起間シナプスの同定の有用性をマウスの嗅球で検討した．まず，嗅球を抗VGLUT1抗体，抗VGAT 抗体で単染色し，電子顕微鏡で観察すると，非対称性シナプス，対称性シナプスのシナプス終末がそれぞれ標識されることが確認できた．次に単一の僧帽細胞をウイルスベクター注入により標識し，抗VGLUT1抗体，抗VGAT 抗体で多重染色して, 僧帽細胞とVGLUT1，VGAT との共存部位を共焦点レーザー顕微鏡で観察した．次いで同部位を電子顕微鏡で同定し，微細構造を解析した．その結果，VGLUT1陽性部位のうち82％に非対称性シナプス，VGAT 陽性部位のうち79% に対称性シナプスが電子顕微鏡で同定でき，VGLUT1，VGAT が，嗅球のシナプスの質的な解析や定量のためのマーカーとして信頼できると結論づけた．また，抗VGLUT1抗体，抗VGAT 抗体で二重染色したものを，高解像度でモンタージュ撮影すると，VGLUT1が外網状層を中心，VGAT が糸球体層に多く分布し，外網状層に中等度分布するなど，新たな知見が得られた．これらの結果は，VGLUT1とVGAT が，シナプスの同定や広範囲の脳領域のシナプスの分布を解析するために有用なマーカーであるということを示している．The olfactory bulb (OB) is the primary center of the olfactory system in the brain. The projection neurons of the OB, mitral cells and tufted cells, form dendro-dendritic synapses with interneurons on their dendrites and relay olfactory information to higher brain regions. Thus, it is necessary to identify the distribution of synapses on mitral cell dendrites to study the processing of olfactory information. High-resolution analysis by electron microscopy (EM) enables morphological identification of synapses; however, examination of large areas is difficult with EM because of its narrow observation range. Further, mitral cells cannot be clearly identified with EM. Vesicular glutamate transporter 1 (VGLUT1) and vesicular GABA transporter (VGAT) are synapse markers and the usefulness of them for estimating synaptic distributions has been reported in many brain regions. Using these markers for quantitative analysis of synapses on dendrites of OB projection neurons may provide higher reliability than EM analysis. In this study, we examined the usefulness of these markers for identification of dendro-dendritic synapses in the mouse OB. First, the OB was single-immunolabeled for VGLUT1 or VGAT and processed for EM. Using EM analysis, we observed VGLUT1 labeling in asymmetrical synapses and VGAT labeling in symmetrical synapses. Second, individual mitral cells were selectively labeled by viral injection and single or double-labeled for VGLUT1, VGAT or both. Labeled sites on mitral cells were analyzed with confocal laser scanning microscopy and subsequently examined with EM. We identified that 82% of VGLUT1-positive and 79% of VGAT-positive sites corresponded to asymmetrical synapses and symmetrical synapses identified by EM, respectively. Thus, we conclude that these synaptic markers are reliable for qualitative observation and quantification of synapses in the OB. Finally, the OB was doublelabeled for VGLUT1 and VGAT, and examined by confocal microscopy with high-resolution montage imaging to analyze distributions of the synaptic markers. VGLUT1 was mainly distributed in the external plexiform layer. VGAT was mainly distributed in the glomerular layer and moderately in the external plexiform layer. These findings indicate that VGLUT1 and VGAT are reliable presynaptic markers for individual synapses and for distribution throughout a brain region

マウス嗅球神経回路におけるセロトニンニューロンのシナプスの微細構造解析

嗅球は明瞭な層構造を持ち,少数のニューロン種から構成され,そこには豊富な化学物質を含むことがわかっており,脳神経回路の解析に有用な領域である.匂い情報を処理する嗅球は,脳の他領域から複数の遠心性ニューロンによる入力を受けており,この一つがセロトニンを含有するニューロン(セロトニンニューロン)である.セロトニンニューロンは,脳全体に広範囲に分布し様々な脳機能の調節を行っており,嗅球においては非対称性シナプスを形成し,嗅覚情報調節に関わっていると考えられている.しかし,このシナプスについては嗅覚調節機構と共に詳細な解析はなされていない.そこで,本研究ではセロトニンニューロンによるシナプスの微細構造を,免疫電子顕微鏡法と電子線トモグラフィーを用いて解析した.また,非対称性シナプスを示すことから,神経伝達物質としてのグルタミン酸の可能性を検討するため,セロトニンとVGLUT3(vesicular glutamate transporter 3)に対する多重蛍光免疫染色法で解析した.セロトニンニューロンによるシナプスは,多くは球形のシナプス小胞を持つが,扁平なものや有心性小胞も存在した.更に,既知のグルタミン酸作動性ニューロンによる非対称性シナプスと比べて,シナプス後肥厚の厚さの多様性が顕著で,シナプス間隙は狭く,シナプスの直径は小さかった.また,セロトニンニューロンの約半数はVGLUT3免疫陽性であり,神経タンパクを含有する有心性小胞を持っていることから,複数の神経伝達物質を含むことが示唆された.シナプス後肥厚は伝達物質であるグルタミン酸の刺激によって厚くなる.セロトニンニューロンは,グルタミン酸を含む複数の神経伝達物質を持つために,グルタミン酸だけを神経伝達物質として持つニューロンが形成する典型的な非対称性シナプスに比べて,多様性のある非対称性シナプスを形成していると考えられる.Because of its simple and distinct layers organized by a few types of neurons and its diverse chemical neuroactive substances, the olfactory bulb (OB) is one of the most desirable regions in which to analyze neuronal organization of the brain. The OB is the primary region that processes odor information and consists of olfactory receptor neurons, projection neurons, interneurons and centrifugal neurons. It is well known that the OB is regulated not only by interneurons but also by centrifugal afferents from other brain regions. Serotonergic fibers derived from the raphe nucleus, one of the centrifugal afferents from other brain regions, the OB is highly innervated by serotonergic fibers. These terminals make asymmetrical synapses onto the target neurons in various synapse formations. However, how different these synapses are from typical asymmetrical and how these differences are related to serotonergic function remains to be clarified. The aim of the present study is to accurately assess the morphometry of the synaptic fine structure of serotonergic neurons as compared with olfactory receptor neurons and projection neurons such as mitral/tufted cells. The synapses were analyzed by pre-embedding immuo-electron microscopy and electron tomography which enables analysis of the synapses in more detail. Additionally, the neurotransmitters of serotonergic neurons were analyzed by immunofluorescence. It was shown that the most common shape of synaptic vesicles of serotonergic fibers was round; the synaptic vesicles of olfactory nerves and dendrites of projection neurons were only round. Synaptic clefts of serotonergic fibers were narrower than that of projection neurons. Postsynaptic density (PSD) of serotonergic synapses was very different from that of olfactory receptor neurons and projection neurons. The diameter of serotonergic synapses, the width of the PSD, was smaller than that of the olfactory receptor neurons. Immunofluorescent study revealed that serotonergic varicosities occasionally co-expressed vesicular glutamate transporter 3 (VGLUT3). This indicates that serotonergic neurons have at least two neurotransmitters, serotonin and glutamate. Dense-core vesicles were characterized as containing monoamines and neuropeptides. In contrast, olfactory receptor neurons and projection neurons, which have been well known to exhibit typical asymmetrical synapses, may have use only one transmitter. PSD is reported to be thickened by stimulation of glutamate. Together with findings of previous studies, the present study suggests that serotonergic synapses might release multiple substances: serotonin, glutamate, and a neuropeptide. Due to these multiple substances, serotonergic neurons may various forms of synapses

成体マウス脳における嗅覚系新生ニューロンの遊走に関する三次元構造解析

哺乳類の嗅覚系ニューロンの一部は生後も新生することが知られている.側脳室前角の脳室下帯で新生した細胞は吻側遊走経路を嗅球に向かって遊走し,嗅球各層のニューロンに分化する.ニューロン新生は再生医学の観点から研究が進んでいるが,神経回路への組み込みなどの分化に関しては未だ不明な点が多い.これは,新生細胞を継時的に追跡した時空間的同定に基づく統合的構造解析の欠如に起因する.そこで本研究は,生後新生するとされる嗅球傍糸球体細胞,顆粒細胞の起源を生体細胞標識法により特定し,標識細胞が遊走し,嗅球に達する過程を正確に同定した後に免疫組織学,電子顕微鏡連続切片三次元再構築,デジタル計測で解析し,新生細胞の遊走と分化の詳細を明らかにすることを目的とした.成体マウス脳室下帯へCell Tracker Orange(CTO)を脳定位的に注入し,1～7日後に灌流固定し矢状断切片を作製,CTO標識された新生細胞が遊走経路に沿って嗅球に到達しているのを確認した.その後,抗CTO抗体を用いた多重免疫染色により経路内の新生細胞の立体構造を同定し,遊走と分化を検討し,また微細構造を透過型電子顕微鏡で解析した.遊走経路のCTO標識細胞はPSA-NCAM陽性で,遊走する新生ニューロンであることを同定した.Neurolucidaによるデジタル形態解析により経路の部位により突起の形態と伸展極性に多様性があることを明らかにした.また嗅球ニューロンのマーカーであるtyrosine hydroxylase(TH)の遺伝子発現が遊走早期に見られる新たな知見をTH-GFPマウスで得た.本研究の生体標識法は遊走と分化の過程を同一標本で確認できる利点があり,遊走しながら形態が変化している.一方,化学的性質を決める遺伝子は,形態変化より前に発現していることがわかり,遺伝子発現と形態の多様性変化について今後の解析課題と言える.In mammals, it is well known that some olfactory neurons are generated in the adult brain. Newly generated cells (NGCs) are continuously born in the subventricular zone (SVZ) at the anterior horn of the lateral ventricle, migrate along the rostral migratory stream (RMS), and differentiate into neurons in olfactory bulb (OB) layers. From the viewpoint of regenerative medicine, adult neurogenesis is attractive and has been investigated by many researchers. It remains to be clarified in detail how these differentiated neurons integrate into the bulbar circuit, however, because few integrative analyses have been done of the structure of NGCs through spatiotemporal identification by tracing during migration from birth to differentiation. Our present study thus aimed to clarify migration and differentiation of NGCs, which differentiate into periglomerular cells and granule cells, by immunohistochemistry, serial-sectioning/reconstruction electron microscopy (serial-EM), and digital morphometry after positive identification of NGCs by vital tracer labeling. First we performed stereotaxic injection of Cell tracker orange (CTO) into the SVZ of adult mice to label NGCs. 1-7days later, fixed brains were cut serially parasagittally. We confirmed CTO-labeled NGCs were distributed and reached the olfactory bulb through the RMS. Thereafter, we identified the threedimensional structure of NGCs, focusing on migration and differentiation using anti-CTO and analyzed ultrastructure by serial-EM. CTO-labeled cells were immunoreactive for polysialylated neural cell adhesion molecule (PSA-NCAM), a well-known marker for migrating NGCs. Digital morphology by Neurolucida indicated new findings, showing structural variability of processes, especially polarity of process extension through the RMS. In addition, we clarified that genetic expression of tyrosine hydroxylase (TH), a marker for bulbar neurons, was found in the early phase after birth using TH-green fluorescent protein (GFP) transgenic mice. The present vital cell-labeling approach we used has the advantage of being able to examine the pleural phase of migration and differentiation in the same section, showing structural changes along with migration. Interestingly, genetic expression was expressed earlier than previously reported. Based on the structural results in the present study, it is worth examining the genetic expression of differentiation that determines the chemical coding of neurons and changes in variability of structure in future projects

Expression analysis of a mouse orthologue of HSFY, a candidate for the azoospermic factor on the human Y chromosome

Heat shock transcription factor on Y (HSFY) is located in one of three candidate regions for azoospermic factor (AZF), AZFb on the Y chromosome. We and others have already revealed that some azoospermic males are missing the regions of the Y chromosome including HSFY. Previously, we showed that murine HSFY-like sequence〔mHSFYL(RikencDNA4933413G11Rik)〕, which is the mouse orthologue of HSFY, is exclusively expressed in testis. The sequences encoding the presumed DNA-binding domain in HSFY and mHSFYL were found in other mammals such as dogs, cows and chickens. To elucidate mHSFYL expression in the testes in detail, we carried out in situ hybridization. mHSFYL was predominantly expressed in round spermatids. Furthermore, we clarified the intracellular distribution of mHSFYL inCOS1 cells with HA- or GFP-tagged proteins. Both HA-mHSFYL and GFP-mHSFYL were located in the nucleus. Our results suggest that HSFY/mHSFYL may have evolutionarily conserved functions for spermatogenesis

Lysophosphatidic acid in medicinal herbs enhances prostaglandin E2 and protects against indomethacin-induced gastric cell damage in vivo and in vitro

Lysophosphatidic acid (LPA) is a bioactive phospholipid that induces diverse biological responses. Recently, we found that LPA ameliorates NSAIDs-induced gastric ulcer in mice. Here, we quantified LPA in 21 medicinal herbs used for treatment of gastrointestinal (GI) disorders. We found that half of them contained LPA at relatively high levels (40–240 μg/g) compared to soybean seed powder (4.6 μg/g), which we previously identified as an LPA-rich food. The LPA in peony (Paeonia lactiflora) root powder is highly concentrated in the lipid fraction that ameliorates indomethacin-induced gastric ulcer in mice. Synthetic 18:1 LPA, peony root LPA and peony root lipid enhanced prostaglandin E2 production in a gastric cancer cell line, MKN74 cells that express LPA2 abundantly. These materials also prevented indomethacin-induced cell death and stimulated the proliferation of MKN74 cells. We found that LPA was present in stomach fluids at 2.4 μM, which is an effective LPA concentration for inducing a cellular response in vitro. These results indicated that LPA is one of the active components of medicinal herbs for the treatment of GI disorder and that orally administered LPA-rich herbs may augment the protective actions of endogenous LPA on gastric mucosa

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

In proliferating neural epithelia, cells undergo interkinetic nuclear migration: stereotyped cell cycle-dependent movements in the apico-basal plane. The microtubule-binding protein Tpx2 is here shown to regulate the G2-phase basal-to-apical migration, while passive displacement effects are responsible for basally directed movements

Olfactory Brain Circuitry : Analysis of the odor deprivation effect using the naris closed mouse model

原著journal articl

Cyclin dependent kinase inhibitorによる神経前駆細胞の分化誘導と脳形成

大脳皮質は知覚,認知,思考等の高次機能を担う中枢である.哺乳類では大脳皮質は6層構造をなし,それぞれの層では機能的,形態的に類似した細胞集団が異なる領域からの投射をやり取りしている.神経細胞およびグリア細胞は,胎生期から生後にかけて神経前駆細胞(Neural progenitor cell,NPC)から分裂し産生される.NPCは時期に応じて神経細胞,続いてグリア細胞へ分化し,最終的な脳の構造が完成する.NPCから分化する細胞運命の決定には,細胞分裂を中止して分化を始める機構が必要であり,その中でもCyclin dependent kinase inhibitors(CKIs)の働きが注目されている.今回我々はこのCKIsのひとつであるp18遺伝子を用いて胎児マウス脳の発生に与える影響を解析した.方法としては胎生13～17日目(E13-17)のマウス胎児脳にp18遺伝子を子宮内エレクトロポレーション法によって強制発現させ,その後に脳組織内での分布を観察した.またCre-LoxP を用いた蛍光蛋白発現システムで個々の細胞形態の立体再構築を行った.その結果,p18遺伝子発現群はコントロール群に比べて多く脳室面側に留まり,特にE14, 15において顕著に認められた.更に遺伝子導入した細胞において分化後の形態を観察したところ,生後脳においてp18遺伝子発現群ではアストロサイトの形態をした細胞が増えており,免疫組織学的染色からもこれらの細胞がアストロサイトであることが示唆された.この細胞はE13～E17いずれの時期においても増加していたが,特にE14,15で顕著に認めたことから,この時期にニューロンの分化からアストロサイトの分化に切り替わる何らかのメカニズムが存在していると考えられた.今回の研究によってNPCがニューロンへの分化からアストロサイトへの分化に切り替わるメカニズムにCKIsが関与している可能性が示唆された.アストロサイトはグルタミントランスポータの発現により,二次的脳損傷から神経細胞を保護しており,その発生が解明されることで低酸素性虚血性脳症の治療への応用も期待される.The cerebral cortex plays a central role in high-level functions such as thinking, perception, and cognition. In mammals, the cerebral cortex has a six-layered structure, and each layer contains cells with similar functions and shapes that exchange signals from different regions of the brain. Neurons and glial cells are produced by differentiation from neural progenitor cells(NPC)from the embryonic stage up to the postnatal stage. NPCs are differentiated into neurons at the embryonic stage, and switch into glial cells at birth, before the structure of the brain is mature. Some molecules have been proposed to determine the cell fate of NPCs by stopping cell division and initiating differentiation. Cyclindependent kinase inhibitors(CKIs)are considered to be important molecular factors in this process. Here we used a CKI, the p18 gene, and analyzed its effect on the formation of the fetal mouse brain. For this purpose, we expressed the p18 gene in the mouse fetal brain at embryonic day 13 to day 17(E13-17), and observed the distribution of the electroporated cells in the cortical tissue. Furthermore, we performed three-dimensional reconstruction of the individual cellular morphology by using the Cre-LoxP fluorescent protein expression system. As a result, the p18-gene expressing cells remained in the ventricular zone more than the control cells. The difference was particularly remarkable at E14 and E15. Importantly, we observed a differentiated cell shape in electroporated cells, and found that the proportion of astrocyte-like cells was greatly increased in the p18 gene-expressing group compared to the control group. We confirmed that these cells were indeed astrocytes with immunohistochemical staining. The number of such astrocyte-like cells increased at any time of electroporation from E13 to E17, especially at E14 and E15. Therefore, we propose that there might be some mechanisms that control the switch from neurons to astrocytes at this stage of development. In conclusion, our study suggests that CKIs are involved in the differentiation program of NPCs from neurons to astrocytes. Astrocytes are known to protect neurons from secondary brain damage by the expression of glutamine transporter. Therefore, elucidating the mechanism of their generation is expected to have applications for the treatment of hypoxic-ischemic encephalopathy in the future