This work was supported by the MND Research Institute of Australia and the Cure for MND Foundation [GIA 1638], the Snow Foundation, and Macquarie University [MQRDG 9201401462]. Imaging was carried out at the Microscopy Facility in the Department of Biomedical Sciences, Macquarie University. Other members of the MND research center are thanked for their contributions to the development of these methods and fish lines.

注:设计，实施和动物实验报告必须采取的现行准则15帐户。这些工作必须事先经当地动物福利机关批准（在本例中，麦考瑞大学的动物伦理委员会）。 1.准备斑马鱼进行安装和UV细胞消融<ol><li>生成斑马鱼（ 斑马鱼 ）表达荧光蛋白。 <ol><li>以表达在斑马鱼兴趣荧光蛋白质中，执行的质粒注射到斑马鱼蛋的单细胞阶段（如别处16中描述），或使用荧光的转基因株系。标记多种类型的细胞，通过杂交确定转基因系相关于感兴趣的问题创造化合物转基因斑马鱼线。放在晚上假底对配合坦克的每侧一雄一雌斑马鱼和光的发病删除分隔第二天早上（如其他地方17详述）。保持斑马鱼在28℃，并根据所建立的协议17,18处理它们。 </li><li>通过使劲穿过塑料滤茶含有胚胎水箱收集成功产卵后的胚胎。冲洗鸡蛋与系统中的水，并将其转移到卵水在培养皿中。 </li><li>检查它们在光学显微镜下，确定施肥。商店受精卵在培养皿中，并放置在孵化器在28°C 18。 </li></ol></li><li>可选:执行注射标记特定的细胞群。 注意:这是一种替代方法，允许表达和蛋白质的可视化，而无需提高稳定的转基因系。这种方法也是有利的，当目的蛋白质是有毒的，并禁止了稳定的反式的代基因线。 <ol><li>注入质粒构建到斑马鱼胚胎的单细胞阶段，如别处19，20，21中描述。 注意:此方法导致感兴趣的蛋白的镶嵌表达。感兴趣的蛋白质是从所选择的启动子驱动的由TOL2侧翼（ 例如，islet1 22 -3mnx1 23，24，满足25，或MPEG1 26）反向重复序列20。 </li></ol></li><li>鱼的年龄到所需的大小。 <ol><li>提高鱼3 - 5天受精后（DPF），并将其放置荧光化合物显微镜下。屏幕的动物进行适当的荧光表达，选择亮标记的鱼。分开适当的幼虫到另一个菜，鸡蛋水嵌入晚R ON（在28℃培养箱店）。 可选:胚胎可以放入在24小时后受精（HPF）0.2毫1-苯基-2- thioures（PTU）林格溶液来抑制色素沉着的形成。必须小心以PTU，因为它是有毒的，并且可以具有不良生理，遗传，或形态影响。 </li><li>为在早期发育阶段的研究（&lt;2旦），dechorionate胚胎使用尖锐的镊子手动。酶通过加入链霉蛋白酶（2毫克/毫升）的蛋水，并在28℃培养他们10分钟Dechorionate大量的胚胎。 </li><li>定期通过胚胎通过塑料巴斯德吸管缓解dechorionation。终止该进程时多数胚胎已经从他们的绒毛膜用鸡蛋水洗他们数次出现。 </li></ol></li><li>准备在琼脂糖斑马鱼嵌入解决方案。 <ol><li>加入4克/升MS222制备麻醉溶液（三卡因原液，pH 7.0）中滴加到含有蛋水的培养皿中。 50 mg / L的剂量是建议起点（ 图1A）。 </li><li>在鸡蛋水 - （1.5％0.8），并将其分装成1.5毫升微量离心管制备低熔点琼脂糖的库存。放置等分试样到预加热的热块（38 - 40℃），并让它平衡到设定的温度（〜30分钟; 图1B）。 </li><li>可选:对于长期成像（&gt; 4小时），准备35毫米的玻璃底培养皿内的小圆圈琼脂糖，并允许它来设置（ 补充图1）。 注:这个额外的步骤能有效地避免在较长的时间框架与斑马鱼全琼脂糖下降的任何移动。 <ol><li>要做到这一点，沿着玻璃底菜的内圆琼脂糖的地方〜300μL准备一个环形圈在中间一个小口，在其中放置鱼（步骤1.5.3; 补充图1） 。 </li></ol></li></ol></li><li>安装在琼脂糖显微镜的斑马鱼。 <ol><li>选择1 - 3预先筛选鱼消融和转移他们（用移液管）与麻醉液菜麻醉幼虫（步骤1.4.1; 图1C;约5分钟）。 注意:鱼时，他们表现出浅鳃盖运动和降低心脏率麻醉，不再显示触摸诱发逃避反应（TEER;未能游开后，轻轻一刷抚摸自己的尾巴）。保证适当的麻醉鱼的道德治疗，防止抽搐时转入琼脂糖或暴露在荧光灯。 </li><li>麻醉被确认后，用一个可调移液管（具有截止200μL的尖端设定为〜30微升）吸起来幼虫并让它沉到尖端的底部。通过释放液态与一滴转移幼虫进入预热琼脂糖（步骤1.4.2）幼虫放入琼脂糖（尽量减少蛋水进入琼脂糖的量; 图1D）。 </li><li>吸上来的鱼琼脂糖包围。很快免除入事先准备好的玻璃底35毫米的菜。 </li><li>使用解剖显微镜和标准油漆刷（长衬垫，大小1）至琼脂糖内的动物的位置上的一侧（头左侧），使主体和尾都是平（ 图1E）。如果有多个鱼工作，对准所有的鱼的菜，让他们使用的共聚焦显微镜以后容易找到。 注:快速进行定位和调整的这个程序（它可能需要一些练习，因为琼脂糖开始暴露在寒冷的温度后立即设置）。 </li><li>离开琼脂糖包埋鱼10 - 15分钟，直到琼脂糖设置牢固。小心补足35毫米的培养皿与含有三卡因〜2蛋毫升水（ 图1F）。 </li></ol></li></ol> 2.设置共聚焦显微镜和成像参数<ol><li>放置在培养皿上共聚焦显微镜阶段嵌入式幼虫并专注于动物脊髓背侧（使用亮场）。检查适当的倍率（40X）和荧光设置下的动物和可视化的感兴趣结构（ 例如，标记的神经元或胶质细胞运动的荧光强度），以确认所有的成像参数是根据需要为后续消融（ 图2）。我们经常使用的40倍物镜来执行我们的时间间隔学习。 </li><li>可选:要进行数小时执行时间推移研究中，最好是记录单个或几个时间点之前消融建立细胞及其环境的未受扰动的生理反应（ 例如，小胶质细胞运动以建立基线速度和运动）。 </li><li>确定结构的厚度为紫外线激光烧蚀兴趣URE。 <ol><li>使用的z驱动器，通过手动聚焦上下验证感兴趣结构（ 例如，细胞胞体）的顶部和底部。记下z平面将要烧蚀（ 例如，小区的中心）。 注:根据经验，这种方法是通过瞄准那名亮标记的脊髓神经元（高信噪比，使消融后易时移可视化; 例如， 图4）最有效和消融的中间胞体。细胞核荧光能的优势，以确保正确的定位和高效率的消融。 </li></ol></li></ol> 3.在斑马鱼脊髓进行单个细胞的有针对性的激光烧蚀注意:对于这个消融和可视化的方法中，使用共聚焦显微镜（莱卡SP5）。使用405纳米二极管细胞特异性DEST消融过程乱子是根据软件（徕卡应用套件，v2.7.3.9723）的详细。然而，配备了405-nm激光和FRAP（荧光漂白后恢复）或漂白模块任何常规共焦显微镜将允许同一小区的操作的性能，但可能具有稍微不同的设置，参数和名称。 <ol><li>通过单击下拉菜单在软件菜单（ 图3A，1和2）的顶端启动FRAP向导。观察具有不同步骤的新窗口，允许所述特定参数为激光烧蚀设置向上（ 图3B，3）。 </li><li>通过选择格式，扫描速度（ 图3B，4），和平均确定消融方法的图像参数（ 图3B中 ，5）。在400赫兹和线的扫描速度1,024×1024的图像格式4平均是最适用的。 注意:通常没有必要改变光谱检测（如激发或发射参数），因为他们已经在先前采集被确定。 <ol><li>如果z平面消融尚未选择（如步骤2.4中所述）中，按在"实时"按钮，并通过试样集中直到荧光结构或将要被消融的所需z平面是焦点。 </li></ol></li><li>一旦通用图像参数设定，访问"漂白"的步骤（ 图3C，6），以控制特定的消融元件。 注意:激光强度的组合（ 图3C，8），扫描速度，并已在步骤3.2（ 图3B，4和5），以及重复将在设置数被设定的平均化步骤3.5（ 图3E，12）中 ，将确定在ROI内的紫外激光的总停留时间，并因此，漂白效率。 <ol><li>通过激活它的漂白过程（ 图3C，8）接合405-nm激光。 注: - 80％在我们的实验装置与上述设定最成功用405-nm激光强度之间60来实现的。要知道，这种激光的输出功率是仪器专用，并会为每一个共焦设置有所不同。 </li><li>使用选项中的"放大"（ 图3C，7）通过减少扫描场，因此最大限度地停留时间在选定的投资回报率最大化漂白力度。另外，使用所选择的用于该方法的软件的"漂白点"选项。 </li></ol></li><li>通过在图像采集风使用任何的绘图工具选择用于烧蚀一个或多个ROI（ 图3D，10）流（ 图3D，9）。定位的轴突岗，例如具有大约4的圆形绘图工具 - 8微米。 注:消融区域是从一个单一的像素，以更大的面积，这取决于应用可调节的。 </li><li>建立ROI之后，选择"时间进程"按钮（ 图3E，11）和确认的周期的感兴趣区将被扫描/烧蚀（ 图3E，12）的数量。以根据需要在漂白过程之后刚刚之前，立即使整个图像的概况选择"预漂白"和"后漂白"帧。 </li><li>建立所有必要的消融参数，按"运行实验"（ 图3E，13）和监测消融后效率。 注:在我们的FRAP设置，单个图像会前，并与合适的激光E中的FRAP周期后拍摄xcitation（ 例如，488纳米的激发对于表达EGFP的细胞）。这些前后消融图片允许的选择消融参数是如何令人满意的投资回报率被漂白的效果如何是一个快速的判断。 </li><li>通过调整激光强度重复这个过程（ 图3C，8），扫描速度和平均化（ 图3B，4和5），并在壳体的重复（ 图3E，12）所选择的投资回报率仍然示出了完成后的高的荧光强度FRAP周期。 </li></ol> 4.执行后续处理，包括鱼"救市"或处置<ol><li>如果实验是终端，安乐死的动物用三卡因的过量。除去蛋水中，并用麻醉原液10分钟更换。为了确保安乐死，检查在显微镜下心跳停止。 </li><li>可选的:如果实验不是终端，从细镊子和刷子琼脂糖小心取出鱼。放置在新鲜鸡蛋水中鱼，并允许它在观察恢复15分钟。如果正常的游泳行为返回，回鱼的孵化器。 </li><li>根据该机构的批准的转基因生物废物流处理转基因动物。 </li></ol>

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(25), (2009).</li><li>Higashijima, S., Hotta, Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+cranial+motor+neurons+in+live+transgenic+zebrafish+expressing+green+fluorescent+protein+under+the+control+of+the+islet-1+promoter/enhancer.">Visualization of cranial motor neurons in live transgenic zebrafish expressing green fluorescent protein under the control of the islet-1 promoter/enhancer.</a> J Neurosci. 20 (1), 206-218 (2000).</li><li>Arkhipova, V., 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=Characterization+and+regulation+of+the+hb9/mnx1+beta-cell+progenitor+specific+enhancer+in+zebrafish.">Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.</a> Dev Biol. 365 (1), 290-302 (2012).</li><li>Flanagan-Steet, H., Fox, M. 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B., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+developmental+model+organism+for+pediatric+research.">Zebrafish as a developmental model organism for pediatric research.</a> Pediatr Res. 64 (5), 470-476 (2008).</li><li>Kabashi, E., 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=Gain+and+loss+of+function+of+ALS-related+mutations+of+TARDBP+(TDP-43)+cause+motor+deficits+in+vivo.">Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo.</a> Hum Mol Genet. 19 (4), 671-683 (2010).</li><li>Laird, A. 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=Progranulin+is+neurotrophic+in+vivo+and+protects+against+a+mutant+TDP-43+induced+axonopathy.">Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy.</a> PLoS One. 5 (10), 13368 (2010).</li><li>Lemmens, 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=Overexpression+of+mutant+superoxide+dismutase+1+causes+a+motor+axonopathy+in+the+zebrafish.">Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish.</a> Hum Mol Genet. 16 (19), 2359-2365 (2007).</li><li>Ramesh, T., 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=A+genetic+model+of+amyotrophic+lateral+sclerosis+in+zebrafish+displays+phenotypic+hallmarks+of+motoneuron+disease.">A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease.</a> Dis Model Mech. 3 (9-10), 652-662 (2010).</li><li>Hanneman, E., Trevarrow, B., Metcalfe, W. K., Kimmel, C. B., 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=Segmental+pattern+of+development+of+the+hindbrain+and+spinal+cord+of+the+zebrafish+embryo.">Segmental pattern of development of the hindbrain and spinal cord of the zebrafish embryo.</a> Development. 103 (1), 49-58 (1988).</li><li>Lewis, K. E., Eisen, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cells+to+circuits:+development+of+the+zebrafish+spinal+cord.">From cells to circuits: development of the zebrafish spinal cord.</a> Prog Neurobiol. 69 (6), 419-449 (2003).</li><li>Ilieva, H., Polymenidou, M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-cell+autonomous+toxicity+in+neurodegenerative+disorders:+ALS+and+beyond.">Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond.</a> J Cell Biol. 187 (6), 761-772 (2009).</li><li>Philips, T., Rothstein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+in+amyotrophic+lateral+sclerosis.">Glial cells in amyotrophic lateral sclerosis.</a> Exp Neurol. 262, 111-120 (2014).</li><li>Mazaheri, F., 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=Distinct+roles+for+BAI1+and+TIM-4+in+the+engulfment+of+dying+neurons+by+microglia.">Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.</a> Nat Commun. 5, 4046 (2014).</li><li>van Ham, T. J., Kokel, D., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cells+are+cleared+by+directional+migration+and+elmo1-+dependent+macrophage+engulfment.">Apoptotic cells are cleared by directional migration and elmo1- dependent macrophage engulfment.</a> Curr Biol. 22 (9), 830-836 (2012).</li><li>Radford, R. 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=The+established+and+emerging+roles+of+astrocytes+and+microglia+in+amyotrophic+lateral+sclerosis+and+frontotemporal+dementia.">The established and emerging roles of astrocytes and microglia in amyotrophic lateral sclerosis and frontotemporal dementia.</a> Front Cell Neurosci. 9, 414 (2015).</li></ol>

The authors declare that they have no competing financial interests.

激光烧蚀途径激光辅助消融技术使细胞的个人或小团体的精确瞄准。结合高分辨率的显微镜和动物模型的遗传操作，如斑马鱼这种技术可以让研究人员系统地研究单个细胞的命运和受伤后的相互作用。 这里所描述的紫外线（405纳米）的激光烧蚀协议概括了单个细胞可以如何强调或杀死选择性（以剂量依赖性的方式），而相邻的神经元，神经胶质细胞，和轴突留下无恙。我们已成功地应用在细胞培养实验这一做法，并在这里描述的斑马鱼脊髓详细的方法。我们通过其它细胞（ 图5，A和网络内选择性地强调个别神经元显示在斑马鱼脊髓这种方法的实施<strong&gt; B），或立即无条件恢复杀死单个神经元（ 图5，C和D）。 先前，专业激光系统，例如脉冲氮激光或双光子激光系统，被要求以诱导组织损伤和运动神经横断10，11，12，13。这些激光系统已成功地使用，以引起细胞损伤，如血栓形成中的动脉和静脉6，急性肾损伤7，心肌损伤8，并研究钙波和脑损伤9后小胶质细胞的反应。此外，苏斯戴尔及其同事使用一种传统的共聚焦装置（351纳米和364纳米紫外激光）来诱导果蝇 14 &lt;损害上皮细胞和神经胶质细胞/ SUP&gt;。 斑马鱼模型的相关性为了解ALS（和其他人类疾病） 斑马鱼是一种广泛使用的模式生物，尤其是对发育研究28，29，30。虽然他们有一定的局限性，其对人类疾病模型，给致病分子机制的认识潜力是巨大的。斑马鱼模型已经很好建立为MND的研究，并已导致重要的分子的见解31，32，33，34。转基因斑马鱼线可以快速产生（4 - 5个月），并允许一个特定的细胞类型，功能，使它们一个有价值的除了ALS的当前动物模型的选择性跟踪。斑马鱼的胚胎/幼虫光学透明，并提供独特的experi精神的优点，允许长期实时成像在大脑或脊髓，不能在啮齿动物模型中容易地实现（或人类）的单细胞水平。当与分子技术，如单细胞消融相结合，这提供了用于在体内研究精确的分子机制的唯一实验平台。 运动神经元可以有选择有针对性的使用紫外激光烧蚀斑马鱼的脊髓神经元开始在出生后10小时内制定并后大约48小时，35，36确立。这种快速发展使得在短的时间框架，并与高通量这些神经元的可视化。运动神经元提供大脑和肌肉之间的重要联系，并在ALS中运动皮层（上位运动神经元），脑干和脊髓（下运动神经元）会受到影响。这些神经元的损失不可避免地导致万亩SCLE萎缩和无力。在斑马鱼的脊髓运动神经元可以通过不同的凸起和由像-3MNX1运动神经元特异性启动子的使用来确定。针对这种投射神经元的胞体显露沿着轴突投射的顺行退化随着时间的推移（ 图4和视频1）。脊髓运动神经元的单细胞分辨率成像还证实激光烧蚀（参见图4和补充视频3参考27）后，磷脂酰丝氨酸易位和随之而来的Annexin V的标签。虽然我们在紫外激光消融方法后死亡的神经元报Annexin V的激活，我们不能确定它是在这个进程加快引发死亡的级联反应，神经退行性疾病或正常细胞的动态平衡过程中发生的神经元死亡完全匹配。 虽然这种消融方法是高度可再现和具体的，不同的嵌入策略也可能影响紫外烧蚀的效率。根据我们的经验，这是最成功的，以我们在嵌入式我们的鱼琼脂糖层最小化。包埋介质与蛋水的附加层可以减少最终由小区接收的紫外线功率由于衰减而发生的散射效应的较厚的层沿着光束路径。 在未来，不同的转基因鱼线的交叉将使对直接和短期的可视化（高达12小时）其他受影响的细胞，如神经胶质细胞的反应，以激光诱导的细胞破坏。例如，星形胶质细胞和非细胞自主毒性在神经变性疾病如ALS一直在研究聚光灯和散发性和家族性ALS 37,38的致病性都在很大程度上牵连。然而，机制底层胶质毒性和选择性对运动神经元仍不清楚。我们和其他人最近采取了这种方法的优点，研究通过小胶质细胞的神经元死去的吞噬和可视残存神经元的27，39，40的间隙。 高分辨率显微术和用于神经炎症标记物相结合的消融技术将允许研究在未来扩展单细胞功能和互连细胞系统的理解。 在体内设置这些过程的描述是不仅在发育的设置，而且在神经变性疾病，包括MND，其中细胞相互作用可能被削弱3，41的模型至关重要。

荧光显微镜长期以来被用于研究在斑马鱼的CNS转基因，特别是其对发展1效应的影响。高分辨率显微术已经允许参与脑发育，肌肉生成，以及许多其他发育事件2的细胞过程的详细映射。研究单个细胞的死亡一直是更具挑战性，主要是由于在标准成像过程中选择性诱导细胞死亡的技术困难。然而，单细胞分辨率成像和高度针对性消融技术的组合允许的应力和损伤直接细胞应答的调查，以及随之而来的细胞 - 细胞相互作用的。理解这些过程是关键的，特别是用于神经变性疾病如运动神经元病（MND），其中神经元的神经胶质细胞的相互作用已经显示出向进展的疾病3。 MND，或肌萎缩性侧索硬化症（ALS），是一种破坏性的神经退行性疾病，影响在脑干，运动皮层，和脊髓运动神经元。这些神经元的丧失导致肌肉松弛，病人在3死- - 5年确诊4。在脊髓链接到肌肉纤维的运动神经元和在促进肌肉收缩中起重要作用。这些神经元的这种交流或死亡的失败逐渐减弱肌肉和影响患者的吞咽，行走，说话，呼吸的能力。可视化运动神经元和短期后果的死亡活的动物提供了极好的机会更好地了解参与正常细胞稳态和疾病的动态过程。 斑马鱼已成为一个有吸引力的模型系统来研究神经退行性疾病1。这个是由于该模型生物体提供，如外部施肥，短发育时间，对神经系统的光接入和易于转基因的优点。此外，为了容易地生成化合物转基因斑马鱼的能力允许对于不同的细胞类型的多个标记策略。遗传消融方法杀死特定的细胞类型可以相当广泛的干扰，但缺乏针对单个细胞5的精细控制。激光辅助的技术，在另一方面，提供优良的时间和空间的控制和已被用于不同的动物模型。尽管大多数方法使用专门的设备，如脉冲激光器6，7，8，9，10，11，12或双光子调校13，其他研究小组最近已采取在常规共焦显微镜14 UV激光的优点。 此处所描述的技术结合高分辨率共焦显微镜用UV激光介导的方法，使细胞应激或死亡中选择的运动神经元的剂量依赖性的方式。它依赖于使用通常安装405纳米的激光，已在细胞培养和活体动物试验成功，并允许细胞相互作用，如神经元死亡后，小胶质细胞间隙的详细特征。

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Agarose low melting</td>
			<td style="width:81px;">Fisher-Scientific</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Dissolve in egg water with tricaine (0.02%) at 0.8-1.5%</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:247px;">Tricaine</td>
			<td style="width:81px;">Argent Labs</td>
			<td style="width:136px;">MS-222</td>
			<td align="right" style="width:191px;">0.02%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Harvard standard wall Borosilicate with filament Capillary Glass</td>
			<td style="width:81px;">World Precision Instruments, Inc.</td>
			<td style="width:136px;">GC100F-10 (short) 
			GC100F-15 (long)</td>
			<td style="width:191px;">Pull to a resistance of 2 -7 M&#937;</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;width:247px;">Egg water&#160;</td>
			<td style="width:81px;"></td>
			<td style="width:136px;"></td>
			<td style="width:191px;">0.6 g Instant Ocean sea salt, 4ml of 1mg/ml methylene blue in 10 l deionized water</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:81px;">Sigma</td>
			<td style="width:136px;">P7629</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Pronase</td>
			<td style="width:81px;">Sigma</td>
			<td align="right" style="width:136px;">10165921001</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">Heat block</td>
			<td style="width:81px;">Select BioProducts</td>
			<td style="width:136px;">Digital block heater (SBD110_)</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:247px;">Microinjection apparatus</td>
			<td style="width:81px;">World Precision Instruments, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Microinjection was performed by hand under a steromicroscope (Leica) with a loaded glass needle and a Picospritzer II (General Valve corporation)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Stereoscope</td>
			<td style="width:81px;">Leica</td>
			<td style="width:136px;"></td>
			<td style="width:191px;">Bright field illumination microscopy was performed on a Leica M165FC stereo dissection microscope (Leica)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Microscope</td>
			<td style="width:81px;">Leica&#160;</td>
			<td style="width:136px;">SP5</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:247px;">35mm glass bottom dish</td>
			<td style="width:81px;">MatTek Corporation</td>
			<td style="width:136px;">P35G-0-20-C</td>
			<td style="width:191px;"></td>
		</tr>
	</tbody>
</table>

triggering cell stress and death using conventional uv laser confocal microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

这里所描述的方法允许运动神经元中使用商业共焦显微镜的FRAP模块斑马鱼脊髓消融。表达在神经细胞绿色荧光蛋白特异性启动子，如控制下的转基因斑马鱼线- 3mnx1，islet1，或满足 ，分别使用。 GFP的由运动神经元的启动子驱动（如-3mnx1或满足 ）的表达使细胞体，主轴突，和外周分支延伸到肌肉（ 图4和视频1）的高分辨率可视化。 在3至5天龄鱼的脊髓神经细胞已经成功地烧蚀，具有60的总停留时间 - 第80号在上述步骤的〜70％的激光功率和一般设置3.成功消融当荧光消融后立即消失实现永不恢复（ 图5，C和D）。在与其他的激光线（如488纳米激光线）消融的尝试并没有导致永久性衰落，和荧光短的时间框架内恢复。重要的是，这种技术显示出在UV-烧蚀神经元的凋亡细胞死亡的特征，如膜联蛋白V的存在，常染色体变性一致的形态变化，并且烧蚀神经元27的轴突起泡。 这种方法的特异性使用photoconvertible荧光团枫（暴露于UV光之后切换从绿色到红色的发射），其中一个已定位的神经元被转化（ 图5，A和B）未经蜂窝迹象的实验证实销毁超过几个小时。较高的激光功率的使用，而不是联To在附近的细胞（〜20微米），以消融部位（ 图5，C和D）的目标神经元（不经光或荧光的再现）和光转换（无死亡）的灭绝。 此激光诱导消融技术的一个重要的优点是该方法的剂量依赖性。定位到不同强度的细胞，微调多层可通过调整激光功率（ 图3C，8），扫描速度和行平均（ 图3B，4和5），ROI的大小待消融（ 图3D，10），以及重复（ 图3E，12）。值得注意的是，这种方法也可用于细胞应激应用于单个细胞而不是诱导细胞死亡。例如，微调已经相当有价值一个神经元的死亡中，以评估细胞过程。长轴突投射是被烧蚀低UV激光强度的运动神经元透露特有的"起泡"（形成和细胞囊泡碎片），它开始在目标胞体和沿轴突随着时间的推移不断（40 - 90分钟; 图4 ; 3D 视频中呈现1本消融的电影）。因此，调制不同激光烧蚀参数和因此引起的细胞应激的水平和死亡的时间过程允许研究的实验灵活性高的水平。 <img alt="图1" src="/files/ftp_upload/54983/54983fig1.jpg" /> 图1:实时成像斑马鱼的嵌入。 （AF）嵌入实时成像步骤:（A）三卡因加入到鸡蛋水，麻醉的斑马鱼<html>的50毫克/升た起始剂量率。 （B）的低熔点琼脂糖（0.8 - 1.5％）的混合物，并加热至38 - 40℃。 （C）使用移液管，在筛选和选择的斑马鱼被转移到用三卡因溶液菜。成功镇静后（浅鳃盖运动，降低心脏速率，缺乏触诱发反应的），一条鱼被转移到预热琼脂糖（D）。最大限度地减少被转移到琼脂糖以防止随后的稀释蛋水的量。 （五）转让琼脂糖一滴-包含斑马鱼到玻璃底35毫米的菜（30〜50微升）。解剖显微镜下进行这一点，并用毛刷轻轻对准斑马鱼的首选方向。等待10 - 15分钟，直到琼脂糖被设置，并添加三卡因溶液〜2毫升的菜（F）。ANK"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54983/54983fig2.jpg" /> 图2:神经元的可视化和小胶质细胞在3-DPF斑马鱼的脊髓。在3天的转基因斑马鱼的脊髓小胶质细胞和神经元的可视化表达（A）GFP阳性神经元（islet1:GFP）和（B）mCherry阳性小胶质细胞（MPEG1:GAL4，UAS:mCherry）。 （C）与亮场图像一起神经元和胶质信道的复合图像。 （C）中的原理插入描绘了鱼的方向，并概述所提出的区域。比例尺= 30微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图3:在紫外激光烧蚀的方法的步骤（如在协议所概述的，步骤3）。步骤控制在共聚焦软件（徕卡应用套件）的FRAP软件模块。 （一）启动FRAP模块进行紫外激光烧蚀的工具。 （B）中设置的z平面像格式，速度和平均烧蚀等FRAP设置，这将决定激光的停留时间。激光强度和"放大"选项，以最大限度地提高效率漂白（三）控制。 （D）的感兴趣区域（ROI）中的一个或多个区域的选择，将被烧蚀。 （E）设置漂白的时间过程确定漂白周期和在感兴趣区的总激光停留时间。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig3large.jpg" target="_blank">请点击她的</a><html> E要查看此图的放大版本。 <img alt="图4" src="/files/ftp_upload/54983/54983fig4.jpg" /> 图4:紫外烧蚀神经元的顺行变性。紫外切除脊髓神经元的神经退行性疾病的时间推移成像。 （AF）单脊髓神经元的紫外线照射（ 满足:GAL4，UAS:EGFP; A;圆）导致神经元萎缩，随着时间的推移围捕（AC）的身体，其次是轴突碎片（CF;箭头） 。在轴突变性开始在SOMA（消融部位），进展顺向轴突远端直到最后，在索玛荧光消失，整个轴突呈"起泡"（DF）。比例尺= 20微米。该消融的3D渲染时间推移电影显示在视频1。ES / ftp_upload / 54983 / 54983fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/54983/54983fig5.jpg" /> 图5:单细胞UV照射的使用在运动神经元一photoconvertible荧光团（枫）的效果的确认。通过photoconvertible荧光枫的一个神经元激活的单细胞的紫外线照射的验证。 （AD）的标有枫神经元的UV照射。 （A）一个单一的神经元（圆）与10秒30％的激光功率的光转化只在个别目标神经元（B）导致的枫光转化（从绿色到红色）。注意，转化细胞存活几个小时，并显示出恶化的无视觉迹象，如起泡或向上舍入。单神经元消融 （C<html> ;圆）具有较高的激光功率（95％对10秒）导致了该神经元（D立即消失）和枫的后续光转化在约20微米的半径范围内的一小部分周围神经元。比例尺= 20微米。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/54983fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="视频1" src="/files/ftp_upload/54983/54983vid1.jpg" /> 视频1:在图4所示的紫外线烧蚀神经元的三维表面再现（了Imaris）。 如图4所示神经元的延时视频是使用可视化软件（了Imaris，位平面）呈现的表面。它突出烧蚀体细胞的收缩过程，随后轴突碎片顺行朝向单元的远端。euron_ablation-3D_rendered.mov"目标="_空白"&gt;请点击这里下载该文件。 补充图1:可选琼脂糖塑像为长时间成像。 为了避免长期采集过程中的鱼类和琼脂糖的运动准备环形沿着玻璃底35mm培养皿（A）的中间边缘琼脂糖形圈。为让〜10分钟琼脂糖集并转移鱼入内圆琼脂糖（B）的下降。尽量减少琼脂糖的量为嵌入（定向后捞出刷过量琼脂糖到外部）。 <a href="http://ecsource.jove.com/files/ftp_upload/54983/Supplementary_Figure_1.jpg" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy at JoVE.com

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

触发细胞应激和死亡使用传统的紫外线激光共聚焦显微镜

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

triggering-cell-stress-death-using-conventional-uv-laser-confocal

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

9.4K 次观看.  Macquarie University. Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

视频: Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

科研

神经科学

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

整个老鼠大脑与光共聚焦显微镜板微米级分辨率光学层析成像

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

神经肌肉接头处:测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

腺相关病毒和共聚焦显微镜对器官脑切片胶质细胞器的可视化和实时成像

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Registration of Calcium Transients in Mouse Neuromuscular Junction with High Temporal Resolution using Confocal Microscopy

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a cascade of events leading to neurotransmitter release. Moreover, changes in presynaptic calcium levels mediate the activity of many intracellular proteins and play an important role in synaptic plasticity. Studying calcium signaling is also important for finding ways to treat neurodegenerative diseases. The neuromuscular junction is a suitable model for studying synaptic plasticity, as it has only one type of neurotransmitter. This article describes the method for loading a calcium-sensitive dye through the cut nerve bundle into the mice's motor nerve endings. This method allows the estimation of all parameters related to intracellular calcium changes, such as basal calcium level and calcium transient. Since the influx of calcium from the cell exterior into the nerve terminals and its binding/unbinding to the calcium-sensitive dye occur within the range of a few milliseconds, a speedy imaging system is required to record these events. Indeed, high-speed cameras are commonly used for the registration of fast calcium changes, but they have low image resolution parameters. The protocol presented here for recording calcium transient allows extremely good spatial-temporal resolution provided by confocal microscopy.

The protocol describes the method of loading a fluorescent calcium dye through the cut nerve into mouse motor nerve terminals. In addition, a unique method for recording fast calcium transients in the peripheral nerve endings using confocal microscopy is presented.

使用共聚焦显微镜以高时间分辨率记录小鼠神经肌肉接头中的钙瞬变

Estimation of the presynaptic calcium level is a key task in studying synaptic transmission since calcium entry into the presynaptic cell triggers a ...