分子，生化学、遺伝学的手法 Molecular, Biochemical, and Genetic Techniques
P1-2-238 単一細胞エレクトロポレーションを用いた培養小脳プルキンエ細胞における遺伝子強制発現 Forced gene expression in cultured cerebellar Purkinje cells using single-cell electroporation ○田中正彦1, 西川心1, 平嶋尚英1 ○Masahiko Tanaka1, Shin Nishikawa1, Naohide Hirashima1 名市大院・薬・生体超分子システム解析学1 Dept Cell Biophys, Grad Sch Pharmaceut Sci, Nagoya City Univ, Nagoya1 The development and function of the CNS are realized through interactions between many cells. To investigate molecular mechanisms of the development and function of the CNS, therefore, it is crucial to be able to manipulate the gene expression of single cells in a complex cell population. Although we recently developed a technique for introducing siRNA into single cells in primary neuronal cultures using single-cell electroporation (Tanaka et al., 2009), we had not succeeded in the forced gene expression using single-cell electroporation. In the present study, we examined several aspects of the experimental conditions to realize the forced gene expression in cultured Purkinje cells using single-cell electroporation. Cerebellar cell cultures were prepared from neonatal mice. Ten to sixteen days in vitro, we placed the tip of a micropipette containing a GFP expression plasmid in contact with the surface of a Purkinje cell and applied square electric pulses with pipette voltages of -2 to -12 V. After that, GFP images of the electroporated cells were captured every day. We altered the internal diameter (I.D.) of glass capillaries for producing micropipettes, the concentration and size of the plasmid, and the pipette voltage of the electric pulses to explore the optimal conditions for the forced gene expression. When we used glass capillaries with the normal I.D. (0.75 mm) for producing micropipettes, the tip of micropipettes was often clogged on electroporation. We solved this problem and succeeded in strong GFP expression in Purkinje cells by using thin-wall glass capillaries with larger I.D. (0.94 mm). Alternatively, by making the size (5.5 kbp) of the original plasmid smaller (3.3 kbp) by digestion, we also succeeded in strong GFP expression in Purkinje cells even when using glass capillaries with the normal I.D. Strong GFP expression in Purkinje cells electroporated under the optimal conditions continued to be observed for more than 20 days after electroporation.	P1-2-239 HiRetとNeuRetウイルスベクターによる逆行性遺伝子導入の特性の比較 Comparison of gene transfer property through retrograde transport between the HiRet and NeuRet vectors ○伊原寛一郎1, 加藤成樹1, 菅原正晃1, 小林憲太2, 小林和人1,3 ○Kanichiro Ihara1, Shigeki Kato1, Masateru Sugawara1, Kenta Kobayashi2, Kazuto Kobayashi1,3 福島医大・医・生体機能1, 生理研・ウイルスベクター開発室2, 科学技術振興機構・CREST3 Dept. Mol. Genet., Fukushima Med. Univ., Fukushima, Japan1, Section of Viral Vector Dev., NIPS, Okazaki, Japan2, JST, CREST3 In order to understand the framework of brain functions, it is important to elucidate the mechanism for the information processing and its regulation in the complex neural circuits. For this purpose, one useful approach is genetic manipulation that controls the activity of specific neuronal types constituting the circuits. We have developed a lentiviral vector showing highly efficient retrograde gene transfer (HiRet) by pseudotyping of the lentiviral vector with fusion glycoprotein B/B2 type, which is composed of parts of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G). In addition, we have created another lentiviral vector for neuron-specific retrograde gene transfer (NeuRet) by pseudotyping with fusion glycoprotein C type, which is consists of a different set of parts of RV-G and VSV-G. These vectors are taken from synaptic terminals and transported through axonal pathway in a retrograde direction, and permits to express the transgene in the neuronal cell bodies localized remote from the injection site. This system makes it possible to manipulate the function of specific neural pathways by injecting some inducible factors into the upstream brain regions. In the present study, we compared gene transfer property in the mouse brain between the HiRet and NeuRet vectors, including the efficiency of retrograde gene transfer into different neural pathways, the time course of retrograde gene transfer after vector injection (1~8 weeks), and the cell type specificity (neuronal and glial cells) around the injected site.
P1-2-240 特定神経路におけるRho GTPaseシグナル伝達系の機能解析 Functional Analysis of Rho GTPase Signaling in Specific Neuronal Pathway ○小林憲太1, 加藤成樹2, 貝淵弘三3, 小林和人2 ○Kenta Kobayashi1, Shigeki Kato2, Kozo Kaibuchi3, Kazuto Kobayashi2 生理研・ウイルスベクター開発室1, 福島医大・生体機能2, 名古屋大院・医・神経情報薬理3 Sup Cen Brain Res, Sec of Viral Vec Dev, NIPS, Japan1, Dept Mol Genet, Inst Biomed Sci, Fukushima Med Univ School of Med, Japan2, Dept Cell Pharmacol, Nagoya Univ Grad School of Med, Japan3 Rho GTPase regulates various neuronal functions, including axon patterning, synapse formation, apoptosis and survival, through some effector proteins. However, the role of Rho GTPase signaling pathway in the brain function remains to be elucidated. Recently, we developed a new gene transfer system, which was combined adeno-associated virus (AAV) vector and highly efficient retrograde gene transfer (NeuRet) vector. We injected NeuRet vector carrying Cre recombinase gene into the striatum of mice (NeuRet-Cre mice), and then injected AAV vector carrying reversed and double floxed-EGFP/RhoA dominant-negative mutant into the medial prefrontal cortex of NeuRet-Cre mice (RhoA DN mice). In RhoA DN mice, the activity of Rho GTPase signaling pathway is suppressed specifically in the medial prefrontal-striatal pathway. Interestingly, RhoA DN mice seemed to exhibit the cognitive impairment in working memory tasks. These results suggest that Rho GTPase signaling plays an important role in the physiological function mediated by the specific neuronal pathway.	P1-2-241 In vivo エレクトロポレーション法を用いた成虫コオロギ脳における遺伝子発現パターンの制御 Control of gene expression pattern within adult cricket brain using in vivo electroporation ○松本‐佐藤千尋1, 設楽久志2, 松田光司3, 中村太郎3, 三戸太郎3, 松本幸久1, 岡浩太郎2, 小川宏人1 ○Chihiro Matsumoto-Sato1, Hisashi Shidara2, Koji Matsuda3, Taro Nakamura3, Taro Mito3, Yukihisa Matsumoto1, Kotaro Oka2, Hiroto Ogawa1 北海道大院・理・生物科学1, 慶大・理工・生命情報2, 徳島大院・ソシオテクノサイエンス・ライフシステム3 Dept Bio Sci, Fac Sci, Hokkaido Univ, Sapporo, Japan1, Dept of Biosci Informatics, Fac of Sci Technol, Keio Univ, Japan2, Dept Life Systems, Inst Tech Sci, Univ of Tokushima, Japan3 Crickets are emerging as a model of hemimetabolous insects in neuroethological research, because of their numerous advantages for studying neural and molecular basis underlying various behaviors, including escape behavior, phonotaxis, mating and associative learning. However, lack of genetic manipulation techniques targeting the nervous system of living adult crickets has been limiting research strategies. Here we developed novel method of efficient gene transfection using electroporation, and report that successful gene delivery was observed in brains of adult crickets (Gryllus bimaculatus) in vivo. In the protocol, we injected plasmid DNA coding a variant of green fluorescent protein (GFP) driven by Gryllus bimaculatus actin (Gb'-act) promoter into the cricket brain, and applied a poring pulse followed by transfer pulses with sufficient voltage levels. We also attempted the local expression targeted to particular brain regions with three strategies; (1) by local injection of DNA (2) by narrowing the electrode positions (3) by fine arrangement of three electrodes. Further, we succeeded in significant expression of genetically-encoded calcium indicator, Yellow Cameleon 3.6 and photo-activated ion channel, Channel Rhodopsin 2, in the cricket brain. Future studies lead by this technique will pave the way to better understanding of the neural systems underlying the cricket behaviors.
P1-2-242 発生過程のマウス脳に対する各AAVセロタイプ（1,2,8,9）およびレンチウィルスの感染特性の比較 Comparative analysis of the tropism and transduction properties of adeno-associated virus 1, 2, 8 and 9 and lentiviral vectors in the developing mouse brain ○大塚正成1, 水上浩明2, 小澤敬也2, 渡我部昭哉1, 山森哲雄1 ○Masanari Ohtsuka1, Hiroaki Mizukami2, Keiya Ozawa2, Akiya Watakabe1, Tetsuo Yamamori1 基生研　脳生物学1, 自治医大 遺伝子治療研究部2 Div. Brain Biology, NIBB, Aichi1, Div. Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical Univ., Tochigi2 Adeno-associated virus (AAV) and lentivirus (LV) are powerful tools for gene delivery for basic neurobiological investigation. To date, gene transfer by AAV vectors has been mostly applied to adult but not so much to fetal brain. In this study, we tested the ability of several AAV serotypes to deliver transgenes to the brain of embryonic mice. We injected AAV vectors (serotype-1, -2, -8, -9) or LV encoding GFP into telencephalic ventricular space of E14 and E16 mouse embryos. The locations of the transduced (GFP-positive) cells in the brains were determined at postnatal 15 days. All the serotypes of AAV transduced both neurons and glia throughout the brain, including the cortex, cerebellum, olfactory bulb and brainstem. AAV8 and 9 had similar transduction profiles, and produced more extensive gene delivery than that by AAV1 and 2. In the cortex, the neurons transduced by each AAV serotype and LV vector were located in different layers ;when each virus was injected at E14, AAV1 and 2 predominantly transduced neurons in layers IV-VI, while AAV8,9 transduced layer VI neurons. On the other hand, LV transduced neurons in layer II-IV. Observation of the cortex two days after injection suggested that differential lamina distribution of these vectors is the result of transduction of neural progenitor cells at different stage of differentiation. These viral vectors should prove highly useful for gain or loss of gene function analysis of neuronal cells belonging to different population in the same brain.	P1-2-243 ラットおよびサル大脳皮質におけるレンチウイルスベクターを用いたプロモーター7種類の比較 Comparison of seven promoters in the rat and monkey cerebral cortices using lentiviral vectors ○矢口雅江1, 大橋陽平1, 坪田匡史1, 佐藤礼奈1,2, 小谷野賢治1, 王寧群1,3, 松山真1, 関根岳1, 宮下保司1,2 ○Masae Yaguchi1, Yohei Ohashi1, Tadashi Tsubota1, Ayana Sato1,2, Kenji W. Koyano1, Ningqun Wang1,3, Makoto Matsuyama1, Takeru Sekine1, Yasushi Miyashita1,2 東京大院・医・統合生理1, 東京大院・理・物理2, 中国首都医科大学　宣武病院3 Department of Physiology, The University of Tokyo School of Medicine, Tokyo, Japan1, Department of Physics, The University of Tokyo School of Science, Tokyo, Japan2, Department of Traditional Chinese Medicine, Xuanwu Hospital of Capital medical University, Beijing, China3 Lentiviral vectors are promising tools that can mediate long-term transgene expression. Although internal promoters are essential for driving gene expression, their properties that affect cell-type specificity and gene expression level are as yet not well known, especially in the nonhuman primate brain. In this study, we compared properties of seven different promoters in vivo, using lentiviral vectors. The seven promoters consisted of five constitutive promoters derived from the murine stem cell virus (MSCV), cytomegalovirus (CMV), CMV early enhancer/chicken beta actin (CAG), human elongation factor-1 alpha (EF1α), and Rous sarcoma virus (RSV), and two cell-type specific promoters derived from rat Synapsin I (Synapsin I) and mouse alpha-calcium/calmodulin-dependent protein kinase II (CaMKIIα). We prepared vesicular stomatitis virus-G (VSV-G) pseudotyped lentiviral vectors expressing EGFP under the control of these promoters and injected them into the rat and monkey cerebral cortices. Immunohistochemical analysis revealed that all the lentiviral vectors have strong endogenous neuronal tropisms in the rat and monkey brains. Among the seven promoters, only the CMV promoter showed significant expression in glial cells (9.4%) in the rat brain, whereas the five constitutive promoters (MSCV, CMV, CAG, EF1α, and RSV) showed expression in glial cells (7.0 - 14.7%) in the monkey brain. The cell-type specific Synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (Synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities to excitatory neurons were lower in the rat brain (Synapsin I, 94.4%; CaMKIIα, 93.9%). These findings would be useful for the design of vectors for delivering transgenes into the rat and monkey brains in the research field of basic and clinical neurosciences.
P1-2-244 ウイルスベクター2重感染法を用いた経路選択的遮断法のマウス指向運動制御系を指標とする考察 Selective blockade of crossed tecto-reticular pathway by double infection of viral vectors impaired orienting responses in mice; methodological considerations ○伊佐かおる1, , 松井亮介3, 加藤成樹4, 木下正治1, 小林憲太5, 渡邉大3, 小林和人4, 伊佐正1,5,6 ○Kaoru Isa1, Thongchai Sooksawate1,2, Ryosuke Matsui3, Shigeki Kato4, Masaharu Kinoshita1, Kenta Kobayashi5, Dai Watanabe3, Kazuto Kobayashi4, Tadashi Isa1,5,6 自然科学研究機構・生理研・認知行動発達1, チュラロンコン大・薬・薬理2, 京都大院・医・生体情報3, 福島県立医大・医・生体機能4, 生理研・ウィルスベクター開発室5, 総研大6 Dept Dev. Phisiol, Nat Inst Phisiol Sci, Okazaki1, Dept Pharm Physiol, Chula Univ, Bangkok, Thailand2, Dept Mol System Biol, Kyoto Univ, Kyoto3, Dep Mol Gen, Inst Biomed Sci, Fukushima Med Scho Med, Fukushima4, Sec Viral Vect Dev, Nat Inst Phisiol Sci, Okazaki5, Dep Life Sci, The Grad Univ Adv St(SOKENDAI), Hayama6 Recently, by using combination of two viral vectors, we developed a technique of pathway-selective and reversible synaptic transmission blockade and succeeded in inducing behavioral deficit of dexterous hand movements by blocking a particular population of spinal interneurons in macaque monkeys. To explore the availability of this technique in other animal species such as rodents, we tried to block the crossed tecto-reticular pathway, which controls orienting responses to visual targets in mice. A neuron-specific retrograde gene transfer vector (NeuRet) carrying the sequences encoding tetracycline responsive element and enhanced tetanus neurotoxin (eTeNT) tagged with green fluorescent protein (GFP) was injected into the left medial ponto-medullary reticular formation. One week later, adeno-associated virus vector with highly efficient Tet-ON sequence, rtTAV16, was injected into the right superior colliculus. Five weeks later, daily administration of doxycycline (Dox) was initiated. One to four days after the Dox administration, orienting responses toward the left side was impaired. Anti-GFP immunohistochemistry revealed that a number of neurons in the intermediate and deep layers of the right superior colliculus were positively stained, indicating expression of eTeNT. After the offset of Dox administration, the anti-GFP staining recovered to the baseline level within 7 days. These findings showed that the pathway-selective synaptic transmission blockade works well as a universal technique with behavioral effects in rodents.	P1-2-245 ロングターゲッティングベクターによるNGL-1ノックアウトマウスの作製 Generation of NGL-1 knockout mice using long targeting vectors ○矢口邦雄1, 岩間瑞穂1, 鈴木瞳1, 斉藤芳之1, 糸原重美1 ○Kunio Yaguchi1, Mizuho Iwama1, Hitomi Suzuki1, Yoshikazu Saito1, Shigeyoshi Itohara1 理研・脳センター・行動遺伝1 Laboratory for Behavioral Genetics, RIKEN BSI, Saitama, Japan1 NGL-1, a synaptic transmembrane protein of the leucine-rich repeat superfamily, mainly localizes to the postsynaptic side of excitatory synapses in the vertebrate brain. NGL-1 protein interacts extracellularly with the presynaptic ligand netrin-G1, and intracellularly with the abundant postsynaptic density protein PSD-95 and other postsynaptic proteins. The netrin-G1/NGL-1 interaction is hypothesized to convey bidirectional intracellular signaling to pre- and post-synapses in a circuit-specific manner and to play a crucial role in higher brain function.To elucidate the function of NGL-1 protein in vivo, we worked extensively to generate an NGL-1 knockout (KO) mice. The use of conventional methods with targeting vectors having homology arms several kilobases in length, however, failed to generate any NGL-1 KO mice. Homologous recombination is a relatively inefficient process, and its efficiency is probably locus-dependent. Because the length of the targeting arms is thought to be a determinant of targeting efficiency, we next used BAC-aided targeting vectors with very long (180~200 kb) homology arms, which led to the successful generation of NGL-1 KO mice. Here we report our method of generating these KO mice using long targeting vectors and the efficiency of this approach.
P1-2-246 フルボキサミンはシグマ1受容体の発現誘導を介して、小胞体ストレスを緩和する Fluvoxamine alleviates ER stress via induction of Sigma-1 receptors (Sig-1Rs) ○近江翼1, 工藤喬1, 谷向仁1, 金山大祐1, 阪上由香子1, 大河内正康1, 原英彰2, 武田雅俊1 ○Tsubasa Omi1, Takashi Kudo1, Hitoshi Tanimukai1, Daisuke Kanayama1, Yukako Sakagami1, Masayasu Okochi1, Hideaki Hara2, Masatoshi Takeda1 大阪大学大学院　医学系研究科　精神医学教室1, 岐阜薬科大学　薬効解析学2 Psychiatry,Univ of Osaka,Osaka1, Department of Biofunctional Evaluation Molecular Pharmacology, Gifu Pharmaceutical University, Japan2 Backgound Recently we showed that Sig-1Rs are transcriptionally induced via the PERK/eIF2α/ATF4 pathway, one of endoplasmic reticulum (ER) stress responses, and ameliorate cell death signaling. Fluvoxamine(Flv) is a widely used antidepressant. Flv behaves as a potent agonist at Sig-1Rs and has the highest affinity of any SSRI. In this study, we investigated effects of flv on the Sig-1Rs induction and ER stress.MethodsWe loaded ER stress on neuroblastoma cell lines with or without flv for LDH assay and western blotting of Sig-1Rs, phosphor-eIF2α, or ATF4. Middle cerebral artery occlusion (MCAO) mice as models for ER stress were also treated with flv for TTC staining to detect infarction. To confirm the Sig-1Rs induction by flv, a luciferase reporter system of its promoter was used. RNA interference was used to knockdown ATF4 expression to check its involvement in the flv effect.ResultsTreatment of neuroblastoma cells with flv reduced cell death induced by ER stress. Paroxetine does not have this effect, which is canceled by NE-100, an antagonist for Sig-1Rs. Intraperitoneal treatment with flv reduced the area of infarction due to MCAO in mice. Flv transcriptionally induces Sig-1Rs bringing cellular resistance to ER stress. In the HEK293 cells with knockdown ATF4 expression, the induction of Sig-1Rs by flv is not observed indicating that this effect of flv is caused by activation of ATF4. Moreover flv does not phosphorylate eIF2α, an overhead of ATF4 expression. Taken together, it is suggested that flv directly activates a promoter of ATF4.ConclusionsFluvoxamine induces Sig-1Rs as a result of direct ATF4 induction by this agent. The induction of Sig-1Rs brings cellular resistance to ER stress. This study shows that flv has the property of alleviating ER stress suggesting that it is a potential drug for diseases caused by ER stress.	P1-2-247 中枢神経系の神経細胞特異的に導入遺伝子が発現する遺伝子組換えマウスの作出と解析 Generation and analysis of transgenic mice that express a transgene specifically in neurons of the CNS ○松崎泰教1, 大上美穂1, 平井宏和1 ○Yasunori Matsuzaki1, Miho Oue1, Hirokazu Hirai1 群馬大学大学院　医学系研究科　神経生理学1 Dept Neurophysiol, Gunma Univ, Gunma1 Promoters being active only in neurons have been used in neuroscience. However, since such cell type-specific promoters are generally much weaker, in terms of promoter strength, than constitutive viral promoters, it is difficult to express sufficient levels of a transgene. Here, using a synapsin I (synI) promoter we aimed to develop lentiviral vectors (LVs) that express a transgene specifically and robustly in neurons. To strengthen the SynI promoter activity, we added the minimal CMV enhancer at the 3' end of the SynI promoter. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was placed following a transgene sequence to increase the stability of the mRNA. The neuronal specificity of the developed LVs was assessed by LV-based generation of transgenic (Tg) mice expressing EGFP. The LVs were infected to 2-cell stage embryos. We obtained 8 Tg mice from 13 pups. All Tg mice showed GFP expression specifically in neurons throughout the CNS. We next generated spinocerebellar ataxia type 1 (SCA1) model mice by insertion of mutant SCA1 gene, ATXN1[Q98], after EGFP. We obtained 6 founder mice, and all 6 mice were genotype positive. Several months after the birth, the Tg mice started to show weight loss and ataxia. Immunohistochemistry revealed that mutant ATXN1 proteins were efficiently and robustly expressed in neurons throughout the CNS. These results suggest that lentiviral vectors carrying the SynI-minCMV promoter plus WPRE is useful for transgene expression robustly and specifically in neurons and for studying pathophysiological roles of neuron-specific genes of unknown function.
P1-2-248 ドーパミン神経でテトラサイクリン遺転子発現誘導システムを作動させるためのマウス Dopamine neuron specific tetracycline-controlled transcriptional activator (tTA) expressing mouse lines ○徐明1, 滝上紘之1, 三村將1, 田中謙二1 ○Ming Xu1, Hiroyuki Takiue1, Masaru Mimura1, Kenji F. Tanaka1 慶應・医・精神科1 Dept. Neuropsychiatry, School of Medicine, Keio University, Tokyo1 Tetracycline-inducible gene expression system (tet system) can be a powerful tool to manipulate a targeted cell function by virtue of forced expressions of functional genes. Tet system requires two distinct lines; one is a cell type-specific tetracycline-controlled transcriptional activator (tTA) expressing line (tTA mouse) and the other is a line with tTA-dependent promoter (tetO) driving expression of gene of interests (tetO mouse). To manipulate dopamine neuron function, dopamine neuron specific tTA line was necessary. Dopamine transporter (DAT) is known to be expressed only in dopamine neuron, thus we generated DAT-tTA bacterial artificial chromosome (BAC) transgenic mouse lines in which tTA was expressed under the control of DAT promoter. We were able to establish 3 different founders, lines 1, 2H, and 15. To examine whether tTA was expressed in dopamine neurons, we performed in situ hybridization for tTA and detected tTA mRNA in dopamine neurons in the substantia nigra and the ventral tegmental area among all lines. We next examined where tTA-mediated gene induction took place by crossing with a sensitive tetO reporter line: tetO-ChR2EYFP knockin. The line 2H exhibited the highest induction in dopamine neurons but ectopic EYFP induction in striatal medium spiny neurons was observed. Although we did not detect any significant level of tTA mRNA in the striatum, a small amount of tTA under the detection level could be able to induce EYFP in the combination with sensitive tetO reporter. Hence, line 1 and 15 exhibited less EYFP induction in dopamine neurons and did not induce EYFP in the striatum. We empirically know that dopamine neurons requires high levels of tTA to successfully induce tetO promoter driving gene expression, therefore, homozygotes of line 1 and 15 might achieve higher penetrance of gene induction in dopamine neurons without ectopic induction.	P1-2-249 腹側線条体間接路神経の選択的除去 Selective elimination of indirect pathway neurons in the ventral striatum ○滝上紘之1,2, 徐明1, 佐野裕美3, 内ヶ島基政4, 渡辺雅彦4, 岡野栄之2, 三村將1, 田中謙二1 ○Hiroyuki Takiue1,2, Ming Xu1, Hiromi Sano3, Motokazu Uchigashima4, Masahiko Watanabe4, Hideyuki Okano2, Masaru Mimura1, Kenji Tanaka1 慶應義塾大・医・精神1, 慶應義塾大・医・生理2, 生理研3, 北大・医・解剖発生4 Dept Neuropsychiatry, School of Medicine, Keio University, Tokyo1, Dept Physiology, School of Medicine, Keio University, Tokyo2, NIPS3, Dept Anatomy and Embriology, Univ of Hokkaido, Hokkaido4 Selective ablation of a specific neuronal population has been utilized to elucidate the complex neuronal circuitry in the striatum. Several types of ablation techniques have been applied so far, however, most techniques required a local delivery of functional molecules and such method inevitably induces surgical injuries and variability of ablation manner between trials. To avoid these disadvantages, we tried to conduct the selective ablation via solely transgenic approach. We first generated a bacterial artificial chromosome (BAC) transgenic mice line in which tetracycline-controlled transcriptional activator (tTA) was expressed under the control of dopamine D2 receptor (D2R) promoter. To confirm where tTA-mediate gene induction was observed, we bred them with ones with the sensitive tTA-dependent promoter (tetO) reporter line: tetO-EYFP knockin and examined EYFP expression profile in double transgenic mice by immunohisotochemistry. We found that EYFP was expressed in D2-MSNs throughout the striatum and we did not detect any immunoreactivity in cholinergic interneurons in the striatum and dopamine neurons in the substantia nigra. We next crossed the D2R-tTA line with tetO-diphtheria toxin A subunit (tetO-DTA mice) to ablate D2R positive cells by virtue of tTA-dependent DTA induction. Unexpectedly the elimination of D2R-MSN was restricted only in the ventral portion of the striatum and the ventral portion selective ablation was consistent between bigenic animals. This transgenic approach permits us to achieve a reliable selective ablation and thus we would be able to address the functional role of ventral D2-MSNs.
P1-2-250 シータ波を利用した海馬CA1領域への選択的遺伝子導入システムの開発と長期増強発現の解析への適用 An application of selective injection system into hippocampus CA1 under monitoring of theta oscillation for analyzing LTP expression ○若園佳彦1, 蔦島譲治1, 國武孝人1, 高宮考悟1 ○Yoshihiko Wakazono1, Jyoji Tsutajima1, Takato Kunitake1, Kogo Takamiya1 宮崎大・医・統合生理1 Dept Neurosci, Univ of Miyazaki, Miyazaki1 Hippocampus is considered to be strongly related to synaptic plasticity function underlying learning and memory, hence, it has been actively investigated for the past several decades. However, their molecular mechanisms remain unclear because of its complexity.The recent cell biological and electrophysiological approaches using dissociated primary cultured neurons is a powerful method to study molecular functions in vitro; however, analysis of intact neuronal circuitry is preferential and more similar to in vivo. One straightforward approach is the direct injection of cDNA/shRNA expressing virus vectors into the hippocampus CA1 pyramidal neurons and analysis of their influence in neuronal functions of normal neuronal circuitry. Most commonly, virus vectors are injected via a pipette that is inserted from the top of the brain cortex. However, it is difficult to accurately locate the tips in mouse CA1 pyramidal cell layer because it is only 0.2mm in thickness. To resolve this problem, we developed a system to accurately inject viral vectors using a stereotactic injection system into the region, with simultaneous electrophysiological monitoring of theta oscillation, and determined the most predictable position of the pipette tip based on the integrated values of theta oscillation at each depth, as reported previously. In the present study, we first tried to transfer exogenous genes, GFP, to hippocampus CA1 by injecting GFP-expressing lentivirus vector. A few weeks later, GFP signals were confirmed in the region and these neurons were able to induce LTP as well as the naive. Next, we tried to transfer an AMPA receptor subunit, GluA1, cDNA into GluA1 knockout mice, which are unable to induce LTP. Preliminary results revealed to rescue LTP expression in the KO mice. This approach provides an efficient method to transfer genes using viral vectors into the hippocampus CA1 neurons in studies of molecular mechanisms of synaptic plasticity and other neuronal functions in vivo.

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