e ポスター

e ポスター 5. 新技術 e Poster 5. New Technology

2020/9/10　14:00～15:00　オンデマンドB-1 P1-32* 培養神経細胞のグルタミン酸刺激で誘発されるドレブリン分布変化の共焦点イメージサイトメーターを用いた定量化アルゴリズムの開発 Optimization of algorithms to quantify changes in drebrin distribution induced by glutamate stimulation of cultured neurons for a confocal quantitative image cytometer 間瀬省吾^1,2、関野祐子¹、白尾智明^2,3、光岡俊成¹ 1. 東京大学大学院薬学系研究科ヒト細胞創薬学寄付講座、2. 群馬大学大学院医学系研究科薬理学、3. アルメッド（株） Shogo Mase^1,2, Yuko Sekino¹, Tomoaki Shirao^2,3, Toshinari Mitsuoka¹ 1. Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, 2. Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Japan, 3. AlzMed, Inc. Activation of NMDA-type glutamate receptors induces drebrin exodus from dendritic spines in matured neurons. We have shown that drebrin immunoreactivity is available for detection of drug effects on synaptic function (Hanamura, et al.2019; Mitsuoka, et al. 2019). In our previous papers, a high-content imaging analysis system based on fluorescence microscopy, IN Cell Analyzer 2200 (GE Healthcare, USA), was used for detecting the loss of drebrin as linear density of drebrin cluster (LDDC) along dendrites. In the present study, we have observed drebrin and MAP2 distribution in neurons with a confocal quantitative image cytometer, CQ1 (Yokogawa, Japan), and developed new algorithms to quantify the glutamate-induced changes in the distribution pattern of their immunoreactivity. Cultured hippocampal neurons (21 DIV) were fixed after glutamate treatments and processed for immunocytochemistry to visualize drebrin with a mouse monoclonal antibody, Map2 with a polyclonal antibody and cell nucleus with DAPI. After automated image acquisitions, total number of drebrin clusters, dendrite length and LDDC automatically quantified with originally developed algorithms. To evaluate the accuracy of our analysis, we used the same preparations which were analyzed with IN Cell Analyzer. The glutamate treatments decreased drebrin cluster density in dose dependent manner as previously reported. We determined one algorithm to quantify the LDDC. In this study, we show that analysis of LDDC is as sensitive enough as the previous system that we used while the algorithms are needed to be optimized for each system.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-33 【誌上発表】赤色蛍光CEPIAを用いたミトコンドリアCa^₂₊動態の可視化解析 Visualization of Ca²⁺ dynamics in mitochondria using red fluorescent CEPIA indicators 金丸和典¹、鈴木純二²、太向勇^1,3、飯野正光¹ 1. 日本大学医学部細胞分子薬理学、2. カルフォルニア大サンフランシスコ校生理学、3. 日本大学医学部生理学 Kazunori Kanemaru¹, Junji Suzuki², Isamu Taiko^1,3, Masamitsu Iino¹ 1. Div. Mol. Cell Pharmacol., Nihon Univ. Sch. Med., 2. Dept. Physiol., UCSF, 3. Dept. Physiol., Nihon Univ. Sch. Med. Mitochondrial Ca²⁺ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca²⁺ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca²⁺ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca²⁺ dynamics, which are assumed to be determined by distinct thresholds of Ca²⁺ increases at each subcellular mitochondrial domain. The balance between Ca²⁺ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca²⁺ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca²⁺ affinities are engineered to match the intramitochondrial Ca²⁺ concentrations. They enable visualization of mitochondrial Ca²⁺ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca²⁺ dynamics and their functions.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-34* オプトジェネティクスを用いた局所脳血流の長期操作と行動変容 A long-term optogenetic manipulation of the local cerebral blood flow in behaving mice 呉雪峰¹、鈴木暢¹、三村将¹、田中謙二¹ 1. 慶應義塾大学 Xuefeng Wu¹, Toru Suzuki¹, Masaru Mimura¹, Kenji Tanaka¹ 1. Keio University Clinical studies have shown a profound reduction of basal ganglia perfusion in patients with apathy, a lack of motivation. We thus construct a hypothesis that the hypoperfusion in the bilateral ventral striatum (VS) attributes to impaired motivation. To this end, we developed a new manipulation technique that enables (1) local, (2) chronic, and (3) reversible change of the cerebral blood flow (CBF) in the mouse brain. We then tested whether the manipulation of local CBF affected the striatum-dependent behavior, e.g., motivated behavior. For the CBF manipulation, we obtained Parvalbumin (Pvalb)-tTA::tetO-channelrhodopsin2 (ChR2) C128S variant-Yellow fluorescent protein (YFP) double transgenic mice (hereafter referred to as PV-ChR2). We demonstrated that the ChR2-YFP only expressed in arterial smooth muscle cells within the striatum in PV-ChR2 mice. Blue light 500 msec illumination to the VS induced a transient reduction of the local CBF, but not resulted in a permanent occlusion. We inserted a dual-LED optic fiber at the bilateral VS and trained the mice in the food-seeking lever press operant task. For the assessment of motivation, mice performed the Fixed Ratio 5 (FR5) task in which mice had to press the rewarded lever five times within 60 seconds. Once behavioral indices on the FR5 task became steady, we started the repetitive optogenetic intervention (0.5 second blue light illumination per hour, 23 times for a day, except for 1 hour task time) and continued for one week. Compared to the baseline indices before the intervention, each PV-ChR2 (n = 2) in the post-one-week optogenetic manipulation showed decreases in successful trial numbers in a daily session and a prolonged 1^st lever press latency from the trial start, reflecting the lowered motivation. Our data indicated that the long-term CBF hypoperfusion mediated by the vascular optogenetics in the striatum caused a decreased motivation in mice.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-35 複数の遺伝子を発現するベクターにおける遺伝子発現リーク機構の解明 Unexpected promoter activity of protein cording sequence in bicistronic constructs confer constitutive gene expression of second gene in bicistronic inducible gene expression vector. 長内康幸^1,2、トバイアスマーソン²、大野伸彦¹ 1. 自治医科大学、2. モナッシュ大学 Yasuyuki Osanai^1,2, Tobias David Merson², Nobuhiko Ohno¹ 1. Jichi Medical University, 2. Monash University Cre-inducible gene expression systems such as lox-STOP-lox cassette is widely used for overexpression of transgenes. Multiple proteins are able to be over-expressed under the transcriptional control of a single promoter by self-cleaving 2A peptide and lox-STOP-lox cassette. Using lox-STOP-lox and P2A peptide, we generated a Cre-responsive bicistronic vector, pCAG-lox-GFP-STOP-lox-rtTA-P2A-mCherry, expressing nucleus targeted GFP in the absence of Cre, and expressing the reverse tetracycline-controlled transactivator (rtTA) plus membrane targeted mCherry after Cre-mediated recombination. Unexpectedly, upon transfection into cells, > 7% of transfected cells misexpressed mCherry without Cre recombination, whereas rtTA was expressed in a strictly Cre-dependent manner. When the order of mCherry and rtTA coding sequences was swapped, mCherry expression was tightly regulated but rtTA expression became leaky (i.e., the second gene is leaky). Changing or removing promoter did not prevent the leaky expression. In silico analysis revealed multiple putative transcription factor binding sites in both mCherry and rtTA CDS. Putting another lox-STOP-lox cassette after 2A sequence dramatically reduced the leaky expression. This study indicates that CDS located 3' region of bicistronic vector with inducible expression construct is leaky because CDS located at the 5' region behave as a promoter. Our data indicate that bicistronic vectors incorporating inducible protein expression cassette always express a small amout of 3' CDS.