光遺伝学的手法 Optogenetics
P3-2-240 マウスfMRIと光遺伝学とを組み合わせた脳活動伝播計測系の構築 Combination of mouse fMRI and optogenetics for measurement of brain activity propagation ○吉田慶多朗1,2, 小牧裕司3,4, 徐明1, 疋島啓吾3,4, 岡野栄之4, 三村將1, 田中謙二1, 高田則雄1 ○Keitaro Yoshida1,2, Yuji Komaki3,4, Ming Xu1, Keigo Hikishima3,4, Hideyuki Okano4, Masaru Mimura1, Kenji Tanaka1, Norio Takata1 慶應義塾大学・医・精神神経科学1, 東京薬科大学・生命科学2, 実験動物中央研究所3, 慶應義塾大学・医・生理学4 Dept Neuropsychiatry, Keio Univ, Tokyo1, Tokyo Univ. Pharm. and Life Sci. Hachiouji, Japan2, Central Institute for Experimental Animals, Kawasaki, Japan3, Dept Physiol, Keio Univ, Tokyo4 Unilateral hippocampus activation via an optogenetic approach resulted in increased locomotion activity with a delay of approximately 100 seconds (Tanaka et al., Cell Rep 2012). Considering immediate firing of hippocampal CA1 pyramidal neurons upon illumination, comprehensive analysis of the manner of activity propagation from the hippocampal region to others should be necessary to understand such delayed behavioral readouts. Although functional magnetic resonance imaging (fMRI) technique could be suited to examine the propagation manner in the whole brain, its application on mouse brains has been limited due to its insufficient spatial resolution. Recently, a method to improve signal-to-noise ratio of fMRI by cooling its probe-coil with Helium gas at ~20 kelvin (called cryoprobe) was reported (Baltes et al., Proc Intl Soc Mag Reson Med 2010), allowing a feasible fMRI application on mice. The drawback of the cryoprobe usage, however, is the lack of the space for optical probe insertion due to its close attachment to the head. To examine how hippocampus-initiating activities propagate, we generated a transgenic mice (Tg-mouse) in which channelrhodopsin (ChR) was selectively expressed in the hippocampal CA1 neurons and developed a method to illuminate the hippocampus under fMRI apparatus with a cryoprobe. We approached the hippocampus from the back of the head; we implanted a 15 mm-fiber optic cannula that went through the cerebellum and the tip of a cannula was directed to the hippocampus. This cannula was able to be attached and detached to a fiber optic patch cable using a cannula mating sleeve, restricting connection of an optical cable to an animal only during fMRI measurement. We succeeded to measure BOLD responses in the hippocampus upon optogenetic manipulation and are currently investigating propagation of the activities.	P3-2-241 HiRetベクターを応用した二重遺伝子導入法による特定神経路におけるチャネルロドプシン遺伝子の高レベルな発現誘導 High level expression of channelrhodopsin gene in specific neural pathways induced by double transduction system with the HiRet vector ○深堀良二1, 加藤成樹1, 小林憲太2, 佐野裕美3, 南部篤3, 八尾寛4, 礒村宜和5, 小林和人1 ○Ryoji Fukabori1, Shigeki Kato1, Kenta Kobayashi2, Hiromi Sano3, Atsushi Nambu3, Hiromu Yawo4, Yoshikazu Isomura5, Kazuto Kobayashi1 福島県立医大・生体機能1, 生理研・ウィルスベクター開発2, 生理研・生体システム3, 東北大・脳機能解析4, 玉川大・脳科学研5 Dept of Mol Genet, Fukushima Med Univ, Fukushima, Japan1, Sec of Viral Vector Development, NIPS, Okazaki, Japan2, Div of Sys Neurophysio, NIPS, Okazaki, Japan3, Lab of Mol and Cel Neurosci, Tohoku Univ, Sendai, Japan4, Brain Sci Institute, Tamagawa Univ, Machida, Japan5 The optogenetic control of neuronal activity helps us to understand the roles of specific neuronal populations in the physiology and behavior. To achieve high level expression of channelrhodopsin (ChR) gene in neural pathways of interest, we developed the double transduction system with a lentiviral vector for highly efficient retrograde gene transfer (HiRet vector) combined with an adeno-associated viral (AAV) vector. We tested the double transduction system mediated by Cre-loxP recombination and tetracycline-dependent transcriptional activation for high level expression of ChR gene. First, the HiRet vector encoding Cre was injected into the dorsal striatum, and then the AAV vector encoding the double-floxed inverted open reading frame construct of a variant of ChR (ChRWR) fused to Venus fluorescent reporter gene (ChRWR/Venus) was injected into the parafascicular thalamic nucleus (PF). In the AAV vector, two type of promoter (CAGGS and EF-1alpha) were used. The ChRWR/Venus expression was observed in the PF, but not in the cortex and the substantial nigra. The intrinsic fluorescence images were also detected in the PF. Second, the HiRet vector encoding ChRWR/Venus under the control of tetracycline response element (TRE) was injected into the striatum, and then the AAV vector encoding tetracycline transactivator (tTA) was injected into the PF. The ChRWR/Venus expression was detected only in the PF. These results show that our double transduction system is useful to induce the high level expression of ChR gene in specific neuronal pathways. This technique will provide a powerful tool to study the function of selective pathways in the neural circuits that mediate a variety of behaviors.
P3-2-242 一次運動野に導入した光感受性膜タンパク質による、行動中のマカクザル前肢筋活動の光遺伝学的抑制 Optogenetic suppression of forelimb muscle activities in the behaving macaque monkey by introduction of microbial opsins in the primary motor cortex ○木下正治1, 笠原洋紀2, 畑中伸彦3, 松井亮介2, 知見聡美3, 伊佐かおる1, 水上浩明4, 小澤敬也4, 南部篤3, 渡辺大2, 伊佐正1 ○Masaharu Kinoshita1, Hironori Kasahara2, Nobuhiko Hatanaka3, Ryosuke Matsui2, Satomi Chiken3, Kaoru Isa1, Hiroaki Mizukami4, Keiya Ozawa4, Atsushi Nambu3, Dai Watanabe2, Tadashi Isa1 生理研・認知行動発達1, 京大院・医・生体情報2, 生理研・生体システム3, 自治医科大・分子病態治療研究セ・遺伝子治療4 Dept Dev Physiol, NIPS, Okazaki1, Grad Sch Biostudies, Kyoto Univ, Kyoto2, Dept System Neurophysiol, NIPS, Okazaki3, Div Genetic Therap, Jichi Medical Univ, Tochigi4 Recently developed optogenetic tools such as channelrhodopsin-2 or halorhodopsin are useful for investigating the functions of neural circuits. Although many optogenetic studies have been reported in small-brained animals such as mice, there are only a few reports about optogenetic modulation of behaviors in non-human primates. In our previous study, we introduced the enhanced halorhodopsin (eNpHR) into the primary motor cortex (M1) of macaque monkeys and showed the optogenetic suppression of spiking activities of M1 neurons under anesthesia. Here we report the optogenetic modulation of forelimb muscle activities in the behaving monkey. In this study, we mapped the M1 regions of a macaque monkey with intracortical microstimulation, and then injected viral vectors carrying hyperpolarizing opsins into the left M1; AAV2-CMV-eNpHR.EYFP into the hand region and AAV2-CaMKIIα-ArchT.GFP into the arm region. Several months after the injections, we photostimulated the injected areas with 561 nm or 589 nm light through the optrode (optical fiber glued with a metal electrode) while the monkey was performing the reach and grasp task. Simlutaneously, we recorded M1 spike activities and electromyogram (EMG) activities of right forelimb muscles. We found the suppression of M1 spiking activities related to the reach and grasp movement and also found the suppression of EMG activities. When we made an electrical stimulation at the same site, we found the opposite effects on EMG activities against to the photostimulation. We showed the optogenetic suppression of EMG activities in macaque monkey, however, reach and grasp behavior itself was not clearly affected. This result implies the possibility of the optogenetics for behavior modulation in a macaque monkey and also implies a necessity of the improvement of this method for applying to the large-brained animals.	P3-2-243 ウィスカ光触覚刺激に対するバレル野の応答解析 Optogenetic excitation of whisker-barrel mechanoreceptive pathway ○本城達也1,2, 住吉晃3, 横山超一1,2, 姫志剛1,2, 川島隆太3, 八尾寛1,2 ○Tatsuya Honjoh1,2, Akira Sumiyoshi3, Yukinobu Yokoyama1,2, Zhi-Gang Ji1,2, Ryuta Kawashima3, Hiromu Yawo1,2 東北大学　生命科学研究科　脳機能解析分野1, 戦略的創造研究推進事業2, 東北大学　加齢医学研究所3 Tohoku Univ. Grad.Sch. Lif Sci, Sendai, Japan1, CREST, JST2, Tohoku Univ. IDAC, Sendai, Japan3 In one of thy1.2-channelrhodopsin 2 (ChR2)-Venus transgenic rat lines, W-TChR2V4, the ChR2 was expressed in the mechanoreceptive subpopulation of trigeminal ganglion neurons which innervate whisker follicles. It is thus well expected that a whisker-related sensory perception should be induced by the photostimulation of their follicles. To test this, the barrel cortex responses were examined using electrophysiological recordings and functional magnetic resonance imaging (fMRI). Under anesthesia with urethane, the whiskers were trimmed and connected with optic fibers of which other endings were connected to LEDs. Pulsative irradiation of blue LED light was used as a test and that of red LED light as control. We found that the blue light irradiation of whisker follicles evoked enhanced unit activities as well as a local field potential in the barrel field of contralateral somatosensory cortex whereas the red light did not. The blue light irradiation also induced blood oxygenation level-dependent (BOLD) and cerebral blood volume (CBV) responses in the barrel field of contralateral somatosensory cortex. It is suggested that the optogenetic whisker stimulation could activate the whisker-barrel cortical pathway of mechanoreceptive signaling. This method would facilitate to study how the spatio-temporal pattern of the whisker mechanoreception would be integrated in the cortex. All animal procedures were conducted in accordance with the guiding principles of Physiological Society of Japan and NIH.
P3-2-244 単一細胞での光遺伝学：ゼブラフィッシュにおいてマウスナー細胞のみを興奮させたときに誘導されるC型逃避行動の解析 Single-cell optogenetics: Activation of a Mauthner neuron in the hindbraininduces C-start escape response in the zebrafish larva ○上萩ちひろ1 ○Chihiro Kamihagi1 兵庫県立大学大学院　生命理学研究科　生命科学専攻1 Grad Sch of Life Science, Univ of Hyogo, Hyogo1 The Mauthner cells (M-cells) are a pair of giant neurons present in thebrain of teleosts and amphibian tadpoles. They are located in the 4thsegment of the hindbrain and one of the most characterized neuronsin vertebrates. They are active during the C-start escape responseand have been considered as a command neuron for this behavior.Physiology, morphology and behavior studies have all indicated tightcorrelation between the M-cell and C-start escape response. Howeverit has not been demonstrated if activation of a M-cell alone can actuallyinduce the behavior. Here, to evaluate this long standing hypothesis, weexpressed the fusion protein of channelrhodopsin 2 and enhanced yellowfluorescent protein (ChR2:EYFP) specifically in a M-cell in zebrafish larvaeby electroporation targeted to single neuron. When ChR2 was expressedspecifically in the left M-cell and the animal was irradiated with blue light,C-start escape was induced towards the right direction. This experimenthas directly proven the command neuron theory for the M-neuron, for thefirst time since its discovery by Mauthner in 1859, and opened up newquestions regarding the single neuron-behavior relation in vertebrates.	P3-2-245 オプトジェネティクスと内因性光信号イメージングを併用したマカクザル皮質間結合パターンの解明 A combination study of optogenetics and optical imaging to identify cortico-cortical projection patterns in macaque monkeys ○中道友1, 橋本光広2, 北村尚士1, 萩谷桂1, 谷藤学1, 佐藤多加之1 ○Yu Nakamichi1, Mitsuhiro Hashimoto2, Naohito Kitamura1, Kei Hagiya1, Manabu Tanifuji1, Takayuki Sato1 理研・BSI1, 名古屋大・医・細胞生物2 Laboratory for Integrative Neural Systems, RIKEN Brain Science Institute, Wako, Japan1, Nagoya University Graduate School of Medicine, Department of Anatomy and Cell Biology, Nagoya, Japan2 Optogenetics has a potential to reveal neural circuits shaping particular response property. However, to identify unknown circuits is still not easy. Suppose that one cortical area (Sender) projects to the other (Receiver). A simple method to identify neural circuits between two areas would be to infect a virus expressing channel rhodopsin in Sender and to detect Receiver activation elicited by Sender optical stimulation. In such approaches, without a priori knowledge about connectivity, it is difficult to determine where to record responses in Receiver. In this study, we explored a possibility to use optical intrinsic signal imaging (OISI) to map activation in Receiver without a priori information. One advantage of using optogenetics for stimulation instead of electrical stimulation is that we may able to specifically stimulate neurons without stimulating passing fibers. To achieve this goal, we used a vector construct of AAV9-CaMKIIa-hChR2(ETTC)-EYFP-MBD, where MBD represents the Myosin Va binding domain of Melanophilin that makes the synthesized protein to be transported to dendrite and soma but not to axons (Lewis, et al., 2009). We also used virus without MBD as control. These viruses were injected into V1/V2 border of one hemisphere (Sender). After several weeks, we exposed the opposite side of V1/V2 border and conducted OISI. We also monitored fluorescence intensity of EYFP at injected surface. We used contralateral V1/V2 border region as Receiver to test feasibility of the combination study of optogenetics and OISI. Monitoring EYFP fluorescence revealed that it reached plateau about 50 days after injection. Anatomical sections revealed axon fluorescence in MBD(-) but only a few axon revealed fluorescence in MBD(+), meaning that expression was limited to cell soma and dendrites in MBD(+). At the injection sites, extracellular recording revealed visual responses, and optical stimulation elicited neural firing. The OISI results will be discussed in the session.
P3-2-246 Optogenetics in freely behaving zebrafish: functional dissection of the habenula ○Felipe A. Fredes1, Ryonoske Amo1, Ryo Aoki1, Mazakasu Agetsuma2, Hitoshi Okamoto1 RIKEN Brain Science Institute1, Dept Biological Sciences, Columbia Univ, New York, USA2 The habenula is a phylogenetically conserved epithalamic center in the vertebrate brain. In mammals, the lateral habenula connects telencephalic nuclei to the brain stem nuclei such as the ventral tegmental area (VTA) and the raphe, and its activation has been demonstrated to be related to negative reward or punishment. The medial habenula in turn, connects to the interpeduncular nucleus (IPN) but its function is still not clear. Our lab previously showed the ventral and dorsal habenula of zebrafish are structurally homologous to the lateral and medial mammalian habenula, respectively. As we have already established the transgenic lines targeting the habenula subnuclei, zebrafish is powerful model system to explore function of the habenula. In order to test the function of the habenular subdivisions independently we have taken advantage of the genetic amenability of zebrafish to express channelrhodopsin-2 (ChR2) specifically in the ventral or dorsal habenula by means of the Gal4-UAS system. Next, to make use of these transgenic animals we have developed the optogenetics stimulation technique for the freely behaving adult zebrafish. By using a micro drill and specially designed micro manipulators, we have succeeded on attaching a 125μm diameter optic fiber to the skull of this animal and keep it stable for two days. With this system we can accurately deliver light stimulation with a 473ηm laser to the habenula with enough power to saturate all the ChR2 molecules. Recently we have designed an avoidance paradigm to test our system. Our preliminary results suggest that animals with a high expression level of ChR2 in the ventral habenula, avoided with a high level of accuracy the stimulation side after the first trials. Control animals showed no levels of preference for either side, with or without stimulation. This new technique can help to understand evolutionary conserved mechanisms of the neural circuits in vertebrate brain.

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