本出版物所报道的研究工作得到国家卫生研究院全国普通医学研究所的支持, R35GM124913、普渡大学 PI4D 奖励计划和 PVM 内部竞争基础研究基金项目。内容完全是作者的责任, 不一定代表供资代理人的官方意见。我们感谢高一川上刚为 Tol2 构造, 林为 ASAP1 构造, 和伦纳德 Zon为ubi 启动子构造通过 Addgene。

斑马鱼被安置在一个 AAALAC 批准的动物设施中, 所有的实验都是根据普渡动物保育和使用委员会 (PACUC) 批准的协议进行的。
1. Tol2 座子质粒结构制备
注: Tol2 是在鳉鱼中发现的座子, 广泛应用于斑马鱼研究社区8,9。它已成功地应用于网关站点特定的基于重组的克隆系统, 并被称为 Tol2 套件10。Tol2 套件允许创建自定义表达式构造的更方便的方法, 同时也提高了转基因的效率。因此, 这是一个简单的决定, 利用这个系统, 并创建一个无处不在的 ASAP1 表达斑马鱼线使用经过验证的泛素启动器来驱动 ASAP111。
<ol><li>创建中间条目 ASAP1 构造: pDONR221-ASAP1<ol>
<li>获得基因编码的电压传感器 ASAP1 构造, pcDNA3.1/普洛-ASAP1 (Plasmid#52519), 从 Addgene。要放大 ASAP1 编码区域, 请使用自定义的引物 (attB1-ASAP1F 和 attB2-ASAP1R) 在底漆的 5 ' 末端 (图 1) 两侧 attB 序列建立 PCR。Phusion DNA 聚合酶的高效性, PCR 条件根据先前发布的协议12进行了优化。</li>
		<li>将50µL PCR 产品装入1% 泰式凝胶中, 使用有200µL 提示的常规吸管, 并在水平凝胶罐中160伏进行电泳, 约30分钟。</li>
		<li>检查在 uv transilluminator (353 nm) 下的凝胶, 用刀片/手术刀在 uv transilluminator 下使用刮刀/刀, 如先前发布的13,14, 并将含有凝胶样品的 DNA 放入干净的1.5 毫升离心管。</li>
		<li>对 ASAP1 PCR 产品进行凝胶纯化。使用商业 DNA 凝胶纯化试剂盒, 在制造商的指导下, 恢复分离凝胶中的 dna, 并将 dna 洗脱到20µL 水中。以1µL 为样本, 用分光光度计测量 DNA 浓度。使用水作为空白控件15流软件指令。</li>
		<li>取100的纯化 PCR 产物, 并将其与 150 pDONR221 质粒在10µL 的 TE 缓冲器 (10 毫米三, 1 毫米 EDTA pH 8.0)16混合。添加2µL 的 BP Clonase II 反应, 并在室温下孵化反应在夜间。</li>
		<li>第二天, 将蛋白酶 K 的1µL 加入反应中, 并在37摄氏度上孵化出30分钟的反应。</li>
		<li>执行转换。转移反应, 并与50µL Top10 能力的大肠杆菌细胞混合, 并在冰上孵化细胞30分钟。然后, 将反应管转移到42°c 水浴中1分钟, 立即取出管并在冰上孵化2分钟。接下来, 把管子放入一个37摄氏度的振动筛中, 孵化1小时。</li>
		<li>取出管子, 将细胞板到卡那霉素 LB 板上。接下来, 在37摄氏度的一夜之间孵化出盘子 (16-18 小时)。</li>
		<li>选择单一和分离良好的殖民地, 并接种到14毫升细胞培养管与3毫升的 LB 培养基。文化他们隔夜在37°c 在一个振动筛以自转速度 250 rpm (自转每分钟)。</li>
		<li>在其指令手册17之后, 使用商业 miniprep 工具包执行 miniprep。</li>
		<li>序列3-4 质粒与 pDONR221-ASAP1 测序, 以识别阳性克隆使用 M13F 和 M13R 序引物。</li>
	</ol>
</li>
	<li>创建用于显微注射的 Tol2 构造: pDestTol2-ubi-ASAP1<ol>
<li>选择已验证 pDONR221-ASAP1 克隆的测序, 并使用水作为空白控件15的软件指令后, 用分光光度计测量其 DNA 浓度。</li>
		<li>采取100µg 的 pDONR221-ASAP1 和混合它与100µg Tol2 套件5端质粒 (pENTR5'_ubi, Addgene #27320), µg p3E-polyA (Tol2 套件 #302) 和150µg pDestTol2pA2 (Tol2 套件 #394)。使用 TE 缓冲器 (10 毫米三, 1 毫米 EDTA, pH 8.0) 在1.5 毫升离心管中调整总容积到8µL, 并由 2-5 s 短涡混合。然后, 添加2µL 的 LR Clonase II 加, 并孵化在室温下的反应过夜。</li>
		<li>第二天, 用10µL 吸管将蛋白酶 K 的1µL 加入反应中, 并在37摄氏度上孵化出30分钟的反应。</li>
		<li>执行转换并标识正克隆, 如上文所述 (步骤 1.1. 7-11)。</li>
		<li>使用分光光度计15测量测序验证的 pDestTol2-ubi-ASAP1 克隆 (图 1B) 的 DNA 浓度。通常集中是大约 200 ng 或µL。</li>
	</ol>
</li>
</ol>2. 制备 Tol2 Transposase mRNA 和注射液
<ol><li>将大肠杆菌甘油 pCS2FA-transposase 质粒 (Tol2 试剂盒 #396) 的股票用灭菌的接种循环放到 LB 板上 (用100µg/毫升氨苄西林)。一夜之间孵化37摄氏度的盘子。第二天, 选择一个单一的殖民地, 并接种它成3毫升磅 (100 µg/毫升氨苄西林) 使用消毒10µL 吸管提示。文化它隔夜在37°c 在一个振动筛以 250 rpm 的自转速度。</li>
	<li>在大肠杆菌区域性上使用商业 miniprep 工具包在其指导手册之后执行 miniprep。用1分钟离心机在 1.4万 rpm 洗脱质粒 dna 30-50 µL, 用分光光度计测定其 dna 浓度。</li>
	<li>进行线性化1-2 µg 质粒与不我限制性和纯化 dna 与 dna 清洁套件后, 它的指导手册后, 我不消化。洗脱将 DNA 转化为5µL 水, 离心1.4万转1分钟。预期浓度约为200-300 µL。</li>
	<li>执行体外转录没有我线性化 pCS2FA-transposase 作为 DNA 模板使用商业 SP6 转录试剂盒。</li>
	<li>一旦反应完成, 纯化 Tol2 transposase mRNA 使用商业 RNA 清洁套件后, 制造商的指示。最后, 将洗脱 mRNA 转化为20µL RNAase 水, 并测定分光光度计中的 RNA 浓度。预期浓度约为1-3 µg/µL. 如果需要, 样品可以储存在-80 摄氏度的冰箱里。</li>
	<li>用 Tol2 在 transposase 管中混合20µL pDestTol2-ubi-ASAP1 和µL 离心 mRNA (100 ng/吹打), 制备微注射溶液。为了防止重复解冻和冷冻引起的核酸降解, 整除6µL 每管, 并将其存储在-80 °c 冷藏库中供将来使用。</li>
</ol>3. 显微注射
<ol><li>在注射前的下午, 设置4-6 个繁殖槽, 至少有2名男性和2名女性。这些鱼不能在下午喂饱。这一步骤将减少鱼的数量和节省时间, 以清洁他们第二天早上, 同时也有助于诱导育种的反应。</li>
	<li>第二天早上, 从-80 °c 冰箱中取出准备好的注射液 (pDestTol2ubi-ASAP1 构造和 Tol2 mRNA), 并将其放在冰上。</li>
	<li>拉在鱼饲养池的分隔, 让鱼交配。一般来说, 鱼在拉出分隔线后20-30 分钟内产卵。如果没有, 再等1-2 小时。有些鱼可能根本不下蛋。在这种情况下, 重复这个实验的鱼胚收集。</li>
	<li>在等待, 确保有针准备与尖端打破了一个角度创建一个斜面边缘与镊子, 或打破一个微妙的任务雨刷。<ol>
<li>使用以下参数在微拉拔上从毛细管玻璃上拔出针头: 热 545;拉 60;速度 80;时间 250;压力500。用镊子在解剖范围 (用眼睛片断标尺为直径估计) 打破针在一个角度近似45°。根据 microinjector 的设置, 所需的针径可以是可变的, 但更小的直径是降低胚胎死亡率的首选。</li>
	</ol>
</li>
	<li>一旦鱼产卵, 收集他们在一个10厘米直径培养皿, 并把他们带到解剖范围。清除所有异常的胚胎和鱼废料。</li>
	<li>将受精的胚胎吸管成3% 琼脂糖注射模。去除多余的水分, 以帮助保持胚胎的到位。</li>
	<li>一旦所有的行都充满了可行的胚胎, 安排它们, 使单个细胞都朝同一个方向向针, 这是关于45°角度水平。这将使注入更容易后。</li>
	<li>戴上手套时, 用20µL 装吸管尖, 从冰管上取出5µL。</li>
	<li>小心地将尖端插入到断毛管的后端, 直到它开始圆锥, 以使试剂接近尖端。如果还有气泡, 摇动针头, 确保不折断笔尖。</li>
	<li>将针头直接插入微注射针夹中, 仔细拧紧, 直到针固定到位。将角度调整为关于45°。</li>
	<li>一旦针准备好并附上, 打开显微镜和气体压力罐。商业 CO2坦克通常是好的为此目的。注射容积由保持和弹射压力调整: 大约 0.5 psi 为持有和 30 psi 为弹射。请务必检查解决方案时, 按下踏板。</li>
	<li>使用一滴矿物油的舞台千分尺, 调整溶液的体积和流量, 以150µm 直径 (约 2 nL)。确保背部压力会让少量滴出针。如果没有足够的背部压力, 毛细管作用将导致液体进入针头和破坏 mRNA。</li>
	<li>一旦针被校准, 开始注射的结构进入受精胚胎的单细胞。 注意: 这需要大量的练习, 耐心和技巧, 因为细胞膜很难刺穿。重要的是, 把溶液注入细胞, 而不是蛋黄, 以生成转基因斑马鱼。这与吗啉代注射液不同。只要结构进入细胞, 针进入细胞的哪一侧就无关紧要了。如果注入蛋黄而不是细胞, 转基因的成功率会很低。单细胞阶段注射也很重要, 或体细胞嵌合体鱼将被创造。这将减少转基因进入生殖细胞的几率。</li>
	<li>使用凝胶缺口的边缘提供一个支持, 保持胚胎到位, 并允许针施加压力, 而不移动胚胎。一旦针的尖端是在单一的细胞, 按下踏板释放所需的解决方案量。对所有的胚胎重复这个过程。</li>
	<li>完成后, 将注入的胚胎通过用鱼系统水和一次性的3.4 毫升转移吸管从琼脂糖槽中冲洗出来, 将注射的胚转移到标记皿中。将胚胎储存在28.5 摄氏度的孵化器中, 让它们发育。在一天内检查清除死鱼胚胎, 并在鱼水中用0.1% 亚甲基蓝代替水。</li>
	<li>注射后大约6-8 小时, 取10个单独注射的鱼胚, 并使用最厉害的方法18制备基因组 DNA。</li>
	<li>第二天早上, 用一个荧光光源解剖显微镜来整理在非蛋黄组织中显示 GFP 的胚胎。这些鱼类胚胎应包含注入结构。</li>
	<li>执行 Tol2 检验以检查座子活动, 如前所述19。如果能检测到切除的质粒, 则保留注入的鱼类胚胎并将其饲养。如果不能检测到切除的质粒, 重复 Tol2 mRNA 的合成和显微注射过程, 直到达到 Tol2 的阳性结果。</li>
</ol>4. 建立转基因 ASAP1 鱼, Tg (ubi: ASAP1)
<ol><li>将注入的鱼 (F0代) 提高到成年, 如斑马鱼书20中所述。这通常需要4月左右。</li>
	<li>采取一个单一的成人 F0鱼和交叉它与单一的性别 wildtype 鱼。在后天繁殖后收集鱼类胚胎。将收集到的鱼类胚胎保存在28.5 摄氏度的鱼水孵化箱中, 0.1% 亚甲基蓝。</li>
	<li>第三天, 用 GFP 过滤器检查荧光解剖显微镜下的鱼胚。分类出绿色鱼类胚胎, 如果有的话, 并提高他们成年为 F1代转基因鱼, Tg (ubi: ASAP1)。 注: 由于大多数父母 F0鱼都是胚芽线遗传合体, 因此不需要孟德尔比。</li>
	<li>交叉单 F1成年鱼与 wildtype 鱼和收集鱼类胚胎。将绿色鱼类的胚胎分类, 并将它们提高到成年时为 F2代鱼。 注: 如果有单一的转基因, 绿色和非绿色鱼类胚胎应接近1:1。</li>
	<li>要查看肿瘤像恶性周围神经鞘瘤 (MPNST) 的电位变化, 交叉 F2代 Tg (ubi: ASAP1) 鱼与rpl35hi258/wt 鱼。众所周知, 可见肿瘤开始在6-8 月大的成人21,22中找到。</li>
</ol>5. 成像
<ol><li>为了图像斑马鱼胚胎, 采取多 F2世代创建者鱼并且横渡他们与 wildtype 鱼在各自的对。根据斑马鱼的分期指南23, 在不同的理想发育阶段收集鱼类胚胎。</li>
	<li>在鱼胚的早期阶段, 在10厘米直径的培养皿中, 用鱼系统水, 在解剖范围内仔细地剥除胚胎的 chorions。</li>
	<li>用3.4 毫升一次性转移吸管将一些鱼类胚胎转移到凹玻璃滑块上, 使用3% 纤维素。将胚胎调整到所需位置, 使用荧光解剖范围下的针来查看蜂窝 GFP 活动。</li>
	<li>对于鱼类胚胎的移动阶段 (较早于 12 somite 阶段), 使用 0.05% tricaine 甲磺酸麻醉鱼胚, 然后将其转移到滑梯上。简单地, 鱼胚在鱼水中被发现成 0.05% tricaine 甲磺酸, 直到它们停止游泳和失去身体平衡。此外, 添加一滴 0.05% tricaine 甲磺酸与纤维素在幻灯片上。</li>
	<li>对于不到 12 somite 阶段的鱼类胚胎, 使用一种免疫荧光复合显微镜与兼容的相机和软件进行成像。对于12岁以上的 somite 阶段鱼类胚胎, 使用荧光解剖显微镜。</li>
	<li>为了图像肿瘤细胞的电压, 首先发现鱼与 MPNST 肿瘤。然后, 麻醉鱼与 0.05% tricaine 甲磺酸。对于整个安装成像, 把鱼变成一个10厘米直径培养皿。为了查看肿瘤细胞的电活动, 在全装成像后可能会解剖出鱼肿瘤。</li>
</ol>

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R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2012).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+plasmid+DNA+from+bacterial+colonies+using+the+QIAGEN+Miniprep+Kit.">Purifying plasmid DNA from bacterial colonies using the QIAGEN Miniprep Kit.</a> Journal of Visualized Experiments. (6), 247 (2007).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), (2007).</li><li>Kawakami, K., Koga, A., Hori, H., Shima, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+of+the+tol2+transposable+element+of+the+medaka+fish,+Oryzias+latipes,+in+zebrafish,+Danio+rerio.">Excision of the tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio.</a> Gene. 225 (1-2), 17-22 (1998).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Amsterdam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+ribosomal+protein+genes+are+cancer+genes+in+zebrafish.">Many ribosomal protein genes are cancer genes in zebrafish.</a> PLoS Biology. 2 (5), E139 (2004).</li><li>Lai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+ribosomal+protein+mutations+are+associated+with+growth+impairment+and+tumor+predisposition+in+zebrafish.">Many ribosomal protein mutations are associated with growth impairment and tumor predisposition in zebrafish.</a> Developmental Dynamics. 238 (1), 76-85 (2009).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics. 203 (3), 253-310 (1995).</li><li>Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+oncogenomic+analysis+of+copy+number+alterations+in+human+and+zebrafish+tumors+enables+cancer+driver+discovery.">Comparative oncogenomic analysis of copy number alterations in human and zebrafish tumors enables cancer driver discovery.</a> PLoS Genetics. 9 (8), e1003734 (2013).</li><li>Zhang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+aneuploid+zebrafish+malignant+peripheral+nerve+sheath+tumors+have+genetic+alterations+similar+to+human+cancers.">Highly aneuploid zebrafish malignant peripheral nerve sheath tumors have genetic alterations similar to human cancers.</a> Proceedings of the National Academy of Science USA. 107 (39), 16940-16945 (2010).</li><li>Urrego, D., Tomczak, A. P., Zahed, F., Stuhmer, W., Pardo, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+channels+in+cell+cycle+and+cell+proliferation.">Potassium channels in cell cycle and cell proliferation.</a> Philosophical Transactions of the Royal Society of London Series B. 369 (1638), 20130094 (2014).</li><li>Yang, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+Imaging+of+Voltage+and+Calcium+Signals+Reveals+Neural+Processing+In+Vivo.">Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo.</a> Cell. 166 (1), 245-257 (2016).</li><li>Chamberland, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+two-photon+imaging+of+subcellular+voltage+dynamics+in+neuronal+tissue+with+genetically+encoded+indicators.">Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.</a> Elife. 6, (2017).</li><li>Hochbaum, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All-optical+electrophysiology+in+mammalian+neurons+using+engineered+microbial+rhodopsins.">All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.</a> Nature Methods. 11 (8), 825-833 (2014).</li><li>Sugiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cell-cycle+progression+in+the+developing+zebrafish+embryo.">Illuminating cell-cycle progression in the developing zebrafish embryo.</a> Proceedings of the National Academy of Science USA. 106 (49), 20812-20817 (2009).</li></ol>

作者没有什么可透露的。

虽然早在胚胎发育和人类疾病中发现了细胞和组织水平的电活动, 但体内动态电变化及其生物学作用仍然很大程度上是未知的。其中一个主要的挑战是可视化和量化的电气变化。膜片钳技术是跟踪单细胞的一种突破, 但是它在脊椎动物胚胎中的应用是有限的, 因为它们是由许多细胞组成的。由于敏感度、特异度和毒性, 目前的化学电压染料也不理想。最近在 GEVIs 发明方面的努力为我们提供了?...

生物电是指由离子通道和位于细胞膜1上的泵所介导的内源电子信号。细胞膜上的离子交换, 以及耦合电位和电流的变化, 对于兴奋性神经细胞和肌肉细胞的信号过程是必不可少的。此外, 生物电和离子梯度也有多种其他重要的生物学功能, 包括能量储存, 生物合成和代谢物运输。生物电信号也被发现作为胚胎模式形成的调节器, 如体轴, 细胞周期和细胞分化的1。因此, 这是至关重要的了解许多人类先天性疾病的结果, 这类信号的错误监管。虽然膜片钳已被广泛用于记录单个细胞, 但它仍然远未理想的同时监测在胚胎发育过程中的多个细胞在体内。此外, 由于其特异性、敏感性和毒性, 电压敏感小分子对于体内应用也不理想。
创建各种基因编码的荧光电压指示器 (GEVIs) 提供了一个新的机制来克服这一问题, 并允许易于应用研究胚胎发育, 即使他们最初是为了监测神经单元格2,3。当前可用的 GEVIs 之一是动作电位 1 (ASAP1)4的加速传感器。它由电压敏感磷酸酶的电压传感域和循环排列绿色荧光蛋白组成。因此, ASAP1 允许可视化的细胞电位变化 (极化: 明亮的绿色; 退极化: 深绿色)。ASAP1 有2毫秒的动力学, 可以跟踪阈的潜在变化4。因此, 这一遗传工具允许在实时生物监测活细胞的新水平的有效性。进一步了解生物电在胚胎发育和许多人类疾病 (如癌症) 中的作用, 将为疾病治疗和预防的关键机制提供新的基础。
斑马鱼已被证明是一个强大的动物模型, 研究发育生物学和人类疾病, 包括癌症5,6。他们分享70% 同源基因与人, 并且他们有相似的脊椎动物生物7。斑马鱼提供相对容易的护理, 大的离合器大小的鸡蛋, 易于处理的遗传学, 容易转基因, 和透明的外部胚胎发育, 这使他们成为一个优越的系统在体内成像5,6。随着大量的突变鱼线来源已经存在和一个完全有序的基因组, 斑马鱼将提供一个相对无限的科学发现范围。
为了研究细胞的体内实时电活动, 我们利用斑马鱼模型系统和 ASAP1。本文介绍了如何利用 Tol2 座子转基因将荧光电压生物传感器 ASAP1 到斑马鱼基因组中, 并在胚胎发育、鱼类幼虫运动和活肿瘤中可视化细胞电活动.

<table><tbody><tr><td>14mL cell culture tubes</td><td>VWR</td><td>60818-725</td><td>E.Coli culture</td></tr><tr><td>Agarose electrophoresis tank</td><td>Thermo Scientific</td><td>Owl B2</td><td>DNA eletrophoresis</td></tr><tr><td>Agarose RA</td><td>Amresco</td><td>N605-500G</td><td>For making the injection gels</td></tr><tr><td>Attb1-ASAP1-F primer</td><td>IDT DNA</td><td>GGGGACAAGTTTGTACAAAAAA&lt;br /&gt;GCAGGCTTCACCATGGAGACGA&lt;br /&gt;CTGTGAGGTATGAACA</td><td>ASAP1 coding region amplification for subcloning</td></tr><tr><td>Attb2-ASAP1-R primer</td><td>IDT DNA</td><td>GGGGACCACTTTGTACAAGAAA&lt;br /&gt;GCTGGGTCTTAGGTTACCACTTC&lt;br /&gt;AAGTTGTTTCTTCTGTGAAGCCA</td><td>ASAP1 coding region amplification for subcloning</td></tr><tr><td>Bright field dissection scope</td><td>Nikon</td><td>SMZ 745</td><td>Dechorionation, microinjection, mounting</td></tr><tr><td>Color camera</td><td>Zeiss</td><td>AxioCam MRc</td><td>Fish embryo image recording</td></tr><tr><td>Concave slide</td><td>VWR</td><td>48336-001</td><td>For holding fish embryos during imaging process</td></tr><tr><td>Disposable transfer pipette 3.4 ml</td><td>Thermo Scientific</td><td>13-711-9AM</td><td>Fish embryos and water transfer</td></tr><tr><td>Endonuclease enzyme, Not I</td><td>NEB</td><td>R0189L</td><td>For linearizing plasmid DNA</td></tr><tr><td>Epifuorescent compound scope</td><td>Zeiss</td><td>Axio Imager.A2</td><td>Fish embryo imaging</td></tr><tr><td>Epifuorescent stereo dissection scope</td><td>Zeiss</td><td>Stereo Discovery.V12</td><td>Fish embryo imaging</td></tr><tr><td>Fluorescent light source</td><td>Lumen dynamics</td><td>X-cite seris 120</td><td>Light source for fluorescence microscopes</td></tr><tr><td>Forceps #5</td><td>WPI</td><td>500342</td><td>Dechorionation and needle breaking</td></tr><tr><td>Gateway BP Clonase II Enzyme mix</td><td>Thermo Scientific</td><td>11789020</td><td>Gateway BP recombination cloning</td></tr><tr><td>Gateway LR Clonase II Plus enzyme</td><td>Thermo Scientific</td><td>12538120</td><td>Gateway LR recombination cloning</td></tr><tr><td>Gel DNA Recovery Kit</td><td>Zymo Research</td><td>D4002</td><td>DNA gel purification</td></tr><tr><td>Loading tip</td><td>Eppendorf</td><td>930001007</td><td>For loading injection solution into capilary needles</td></tr><tr><td>Methylcellulose (1600cPs)</td><td>Alfa Aesar</td><td>43146</td><td>Fish embryo mounting</td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td>Suppresses fungal outbreaks in Petri dishes</td></tr><tr><td>Microinjection mold</td><td>Adaptive Science Tools</td><td>TU-1</td><td>To prepare agaorse mold tray for holding fish embryos during injection</td></tr><tr><td>Microinjector</td><td>WPI</td><td>Pneumatic Picopump PV820</td><td>Microinjection injector</td></tr><tr><td>Micro-manipulator</td><td>WPI</td><td>Microinjector MM3301R</td><td>Microinjection operation</td></tr><tr><td>Micropipette puller</td><td>Sutter instrument</td><td>P-1000</td><td>For preparing capillary needle</td></tr><tr><td>Mineral oil</td><td>Amresco</td><td>J217-500ml</td><td>For calibrating injection volume</td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription Kit</td><td>Thermo Scientific</td><td>AM1340</td><td>mRNA in vitro transcription</td></tr><tr><td>Monocolor camera</td><td>Zeiss</td><td>AxioCam MRm</td><td>Fish embryo image recording</td></tr><tr><td>Plasmid Miniprep Kit</td><td>Zymo Research</td><td>D4020</td><td>Prepare small amount of plasmid DNA</td></tr><tr><td>Plastic Petri dishes</td><td>VWR</td><td>25384-088</td><td>For holding fish or fish embryos during imaging process</td></tr><tr><td>RNA Clean &amp;amp; Concentrator-5</td><td>Zymo Research</td><td>R1015</td><td>mRNA cleaning after in vitro transcription</td></tr><tr><td>Spectrophotometer</td><td>Thermo Scientific</td><td>NanoDrop 2000</td><td>For measuring DNA and RNA concentrations</td></tr><tr><td>Stage Micrometer</td><td>Am Scope</td><td>MR100</td><td>Microinjection volume calibration</td></tr><tr><td>Thermocycler</td><td>Bio-Rad</td><td>T100</td><td>DNA amplification for gene cloning</td></tr><tr><td>Thin wall glass capillaries</td><td>WPI</td><td>TW100F-4</td><td>Raw glass for making cappilary needle</td></tr><tr><td>Tol2-exL1 primer</td><td>IDT DNA</td><td>GCACAACACCAGAAATGCCCTC</td><td>Tol2 excise assay</td></tr><tr><td>Tol2-exR primer</td><td>IDT DNA</td><td>ACCCTCACTAAAGGGAACAAAAG</td><td>Tol2 excise assay</td></tr><tr><td>TOP10 Chemically Competent E. coli</td><td>Thermo Scientific</td><td>C404006</td><td>Used for transformation during gene cloning</td></tr><tr><td>Tricaine mesylate</td><td>Sigma-Aldrich</td><td>A5040</td><td>For anesthetizing fish or fish embryos</td></tr><tr><td>UV trans-illuminator 302nm</td><td>UVP</td><td>M-20V</td><td>DNA visualization</td></tr><tr><td>Water bath</td><td>Thermo Scientific</td><td>2853</td><td>For transformation process of gene cloning</td></tr></tbody></table>

visualization of cellular electrical activity in zebrafish early embryos and tumors

生物电, 由离子通道和细胞膜上的泵介导的内源电信号, 在兴奋性神经细胞和肌肉细胞的信号过程和其他许多生物学过程中起着重要作用, 如胚胎发展模式。但是, 在脊椎动物胚胎发生中需要进行体内电活动监测。基因编码的荧光电压指示器 (GEVIs) 的发展, 使我们有可能为这一挑战提供解决方案。在这里, 我们描述如何创建一个转基因的电压指示器斑马鱼使用已建立的电压指示器, ASAP1 (动作电位加速传感器 1), 作为一个例子。本研究选择了 Tol2 套件和无处不在的斑马鱼启动子ubi 。我们还解释了网关站点特定克隆的过程, Tol2 座子斑马鱼转基因, 以及早期的鱼类胚胎和鱼类肿瘤的成像过程, 使用常规 epifluorescent 显微镜。利用这条鱼线, 我们发现在斑马鱼胚胎发生和鱼类幼虫运动过程中存在着细胞电压的变化。此外, 据观察, 在一些斑马鱼恶性周围神经鞘肿瘤, 肿瘤细胞一般极化相比, 周围正常组织。

在一个成功的注射, 超过50% 注入鱼类胚胎将显示一定程度的绿色荧光在体细胞, 其中大部分将是积极的 Tol2 座子的消费检测 (图 2)。经过2-4 代与 wildtype 鱼的杂交 (直到荧光鱼达到 50%, 预期的孟德尔比), 转基因鱼被用于成像实验, 以跟踪细胞膜电位在胚胎发育过程中。首先, 在斑马鱼早期胚胎发育阶段, 在整个细胞周期内检查膜电位的变化。据观察, 细胞...

Watch this Scientific Journal Video about Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors at JoVE.com

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

在这里, 我们展示了创建一个细胞电压报告斑马鱼线的过程, 以可视化胚胎发育, 运动和鱼类肿瘤细胞在体内。

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

visualization-cellular-electrical-activity-zebrafish-early-embryos

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.7K 次观看.  Purdue University. 在这里, 我们展示了创建一个细胞电压报告斑马鱼线的过程, 以可视化胚胎发育, 运动和鱼类肿瘤细胞在体内。

视频: Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

科研

神经科学

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

发育生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

斑马鱼幼虫的前脑的电生理记录

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼:可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...