Name: immobilization of caenorhabditis elegans to analyze intracellular transport in neurons
Uploaded: 2017-10-18T15:00:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

作者对 Dr. 麻子杉本 (东北大学) 的有益讨论深表谢意。wyIs251是 Dr. 康沈 (斯坦福大学) 的一份慷慨礼物。mnIs17由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 出资。这项工作得到了 jsp KAKENHI 赠款 #17H05010 和 #16H06536 和第一三基金会, 脑科学基金会和内基金会的支持。

1. 样品制备 <ol> <li> 使用转基因方法生成转基因的 线虫的兴趣 18 。或者, 从 线虫 遗传学中心获得合适的菌株 (CGC)。为了可视化突触囊泡前体的轴突转运, 请使用转基因线 wyIs251 [ Pmig-13::gfp::rab-3 ] 7 。要观察 IFT, 获得转基因线 mnIs17 [ osm-6::gfp ] 19 。这些菌株在本协议中使用. 
	注: 无论是外阵列, 基因组整合, 甚至 GFP 基因作品。要减少背景信号, 请使用特定于细胞的启动子, 而不是泛神经启动子. 
	注意: 蠕虫的维护在其他协议 20 中有说明。在线虫生长培养基 (NGM) 板 (1.7% (w/v) 琼脂糖上, 在 Escherischia 大肠杆菌 OP50 饲养者的草坪上维持菌株. 50mM 氯化钠, 0.25% (含钒) 蛋白胨, 1 毫米 CaCl 2 , 5 毫克/毫升胆固醇, 25 mm, 2 PO 4 , 1 mm MgSO 4 在 60 mm 直径的塑料碟上) 在20和 #176; C. </li>
	<li> 在20和 #176 中生长蠕虫; C 在 NGM 板上, 到蠕虫的任何生命周期阶段. 
	注意: 在任何阶段都可以观察轴突运输和 IFT。晚 L4 对年轻成人是一个很好的积极控制, 因为多个出版物使用了这些阶段和可视化的贩运活动 14 , 21 , 22 , 23 . </li>
</ol> 2。10% 琼脂糖的制备 注意: 许多供应商提供类似的产品, 如琼脂、琼脂粉和琼脂糖。使用电泳级琼脂糖 (凝胶强度和 #62; 1200 克/厘米 2 )。廉价的琼脂粉不起作用, 因为产生的凝胶不够坚固, 无法固定蠕虫. 
<ol> <li> 在玻璃管 (1.5 cm x 10.5 厘米) 的4毫升蒸馏水 (DW) 中混合0.4 克琼脂糖。在玻璃管上盖上盖子以避免蒸发. </li>
	<li> 将玻璃管放在95和 #176 上; C 热块90分钟。此外, 准备另一玻璃管 (1.5 厘米 x 10.5 厘米) 填充 DW, 并保持在95和 #176; c. 在95和 #176 中放置一个巴斯德吸管; c DW ( 图 1 a )。在琼脂糖管中会出现很多小气泡, 溶液混浊。离开它, 直到解决方案变得清晰。然后, 移混合, 直到琼脂糖溶液变得均匀. 
	注: 微波不能用于制备10% 琼脂糖溶液时, 由 DW 和琼脂糖。微波导致的小气泡阻止了琼脂糖的融化。然而, 固化10% 琼脂糖凝胶可以很容易地融化再次使用微波。含有10% 琼脂糖凝胶的玻璃管可以提前准备, 储存在4和 #176; C 至少6月。如果这样做, 在需要时用微波炉熔化库存, 并从本协议的步骤3开始. 
	注: 琼脂糖在95和 #176 时逐渐失去凝固的能力; C 长时间或反复凝固和熔化后。如果发生这种情况, 准备新的10% 琼脂糖. </li>
</ol> 3。琼脂糖垫的制备 <ol> <li> 使用在步骤2.2 中准备的预热的巴斯德吸管, 在 76 x 22 mm 幻灯片上放置2滴10% 琼脂糖. </li>
	<li> 快速覆盖第二个 76 x 22 mm 2 在琼脂糖滴凝固之前滑动, 如图 1 B 所示, 然后向下推第二张幻灯片以拼合琼脂糖溶液。厚度应为 0.5-1 mm. </li>
	<li> 快速冲洗移95和 #176 的巴斯德吸管; C DW 向上和向下直到琼脂糖溶液被洗掉。否则, 吸管会被琼脂糖凝胶堵塞。将吸管放在95和 #176; C DW. </li>
	<li> 等待1分钟。琼脂糖垫形成并且变得多云。然后, 手动滑动幻灯片 ( 图 1 C ). </li>
</ol> 4。安装蠕虫 注意: 即使是少量的蠕虫运动也会阻碍良好的观察。左旋咪唑传统上用于防止在琼脂糖垫 24 , 25 上的蠕虫移动。然而, 左旋咪唑在 C. 线虫 中抑制神经元受体, 因此可能影响神经元中的贩运事件 15 。使用聚苯乙烯微描述存在一个好的替代 17 . 
<ol> <li> 在装有可镜像透视的立体声显微镜下, 将30转基因蠕虫转移到一个新的 NGM 板上, 用铂丝镐来表示不带细菌的适当标记. 
	注: 白金线镐是由铂丝和巴斯德吸管 (5 英寸), 铂金丝的尖端被夷为平地。在 Wormbook (http://www.wormbook.org) 中描述了铂丝采摘和处理这些蠕虫的准备工作. </li>
	<li> 离开盘子1分钟. 细菌会自动从蠕虫的表面移除, 因为蠕虫在无细菌的 NGM 琼脂糖凝胶上移动. </li>
	<li> 将 0.5-1.0 和 #181; 将 2.6% 100 nm 直径的聚苯乙烯微在水中放在琼脂垫上 ( 图 1 D )。将10-20 蠕虫从无菌 NGM 板转移到微。使用铂线拾取来除去和传播蠕虫, 以避免蠕虫体的重叠 ( 图 1 E ). </li>
	<li> 应用 22 x 40 mm 2 片 ( 图 1 F )。轻轻推, 以消除气泡, 但避免粉碎蠕虫。该示例现在已准备好进行成像. 
	注: 由于不进行麻醉, 蠕虫只由琼脂垫、聚苯乙烯微和片 17 产生的摩擦力来固定。饲养细菌的存在和/或过多的溶液会使摩擦力减弱. 
	注意: 不同大小的片 ( 例如 , 22 x 22 mm 2 , 18 x 18 mm 2 ) 工作。然而, 更大的片可以防止浸入水或油从物镜泄漏到样品期间的观察. </li>
</ol> 5。观察 注意: 对于每个显微镜系统和照相机, 适当的成像参数 (激光功率、增益、分、 等 ) 将有所不同。在这里, 一个 widefield 显微镜配备了旋转磁盘共焦扫描仪和数字 CCD 相机使用. 
<ol> <li> 将舞台温度控制设置为20和 #176; c, 或将室温调整为20和 #176; c. </li>
	<li> 将样本幻灯片放在显微镜阶段。使用 100x (数值孔径 = 1.3 或更好) 物镜. </li>
	<li> 查找 图 2 A (用于 wyIs251 和轴突传输) 或 图 2 B (用于 mnIs17 和 IFT) 作为正控制的区域.可以分析其他区域以及 22 . </li>
	<li> 设置参数 (CCD 增益 = 200, 分 = 1 或 2, 激光功率在 488 nm = 1.0-1.3 兆瓦)。执行4帧/秒的时间间隔记录, 最多1分钟将文件保存为多-TIFF 格式. 
	注意: 如果 GFP 信号消失, 很难继续观察 GFP::RAB-3 或 OSM-6::GFP 三十年代, 激光功率太强。在这种情况下, 减少激光功率。反之, 如果没有记录水泡运动, 激光功率太弱。在这种情况下, 增加激光功率. </li>
	<li> 观察不同的蠕虫并重复5.3 和5.4。观察可以持续多达20分钟, 但每个蠕虫应该只记录一次, 以避免影响 photo 损坏. 
	注意: 蠕虫已被观察至少20分钟没有明显的缺陷 7 , 27 , 28 。更长的观察也许是可能的, 但避风港和 #39; 没有被测试。神经元死亡的一个明显的迹象是神经元形态的变化, 如突起肿胀。由于弱 GFP 信号可以看到在轴突和树突在两个 wyIs251 和 mnIs17 , 突起形态学可以很容易地检查只看在轴突或树突。神经元形态学在 图 2 中显示. </li>
	<li> 生成影片文件. 
	<ol> <li> 使用斐济软件打开多 TIFF 文件。转到文件和 #62; 打开然后选择在步骤5.4 中保存的多 TIFF 文件. 
		注: 斐济可下载 jttps://斐济。图像 J 和插件也可以用来生成 kymographs。如果这样做, 请按照所选插件的相关说明进行操作. #160; </li> <li> 单击 #34; 矩形选择和 #34; 按钮 ( 图 3 , 箭头), 并通过绘制矩形来选择感兴趣的区域使用计算机鼠标 ( 图 3 a 中的黄色矩形)。转到图像和 #62; 堆栈和 #62; 工具和 #62; 使 Substacks 并选择影片中包含的切片编号。生成 substack. </li>
		<li> 通过单击并转到图像和 #62 来激活 substack 窗口; 调整和 #62; 亮度/对比度。一个调整亮度和对比度的窗口出现了。在窗口中, 将顶部 (最小) 向右滑动, 使背景信号消失, 第二个顶栏 (最大) 向左, 以便在背景下可以看到移动的泡和颗粒. </li>
		<li> 转到图像和 #62; 堆栈和 #62; 时间压模。由于影片在步骤5.4 的4帧/秒录制, 时间间隔为0.25 秒。因此, 类型和 #34; 0.25 和 #34; 间隔. </li>
		<li> 从 "文件" 菜单 (影片1和 2) 中选择 "保存为" 和 "#62", 然后生成影片文件. 
		注意: 通过使用适当的插件, 您可以将文件保存为其他格式, 如和 #34; mpeg4 和 #34; 或 #34; #34; 文件. </li>
	</ol> </li> <li> 生成 kymographs. 
	<ol> <li> 再次使用斐济软件打开原始影片文件, 并重复步骤5.6.2 和5.6.3 以调整亮度和对比度. </li>
		<li> 单击和 #34; 分段行和 #34; ( 图 3 B , 箭头)。使用鼠标, 绘制沿纤毛或轴突 (黄线, 在 图 3 B ) 的分段线. </li>
		<li> 转到分析和 #62; 多 Kymograph 和 #62; 多 Kymograph ( 图 3 C )。将出现 "线条宽度" 窗口 ( 图 3 D )。类型和 #34; 3 和 #34; 在 "线宽" 窗口中, 单击 "确定" ( 图 3 D ). </li>
		<li> Kymograph 窗口已创建。以 TIFF 或 JPEG 格式保存图像. </li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hirokawa, N., Niwa, S., Tanaka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+motors+in+neurons:+transport+mechanisms+and+roles+in+brain+function,+development,+and+disease.">Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease.</a> Neuron. 68 (4), 610-638 (2010).</li><li>Holzbaur, E. L., Scherer, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubules,+axonal+transport,+and+neuropathy.">Microtubules, axonal transport, and neuropathy.</a> N Engl J Med. 365 (24), 2330-2332 (2011).</li><li>Kamiya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+diversity+of+axonemal+dyneins+as+studied+in+Chlamydomonas+mutants.">Functional diversity of axonemal dyneins as studied in Chlamydomonas mutants.</a> Int Rev Cytol. 219, 115-155 (2002).</li><li>Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport+motors+in+cilia:+moving+along+the+cell's+antenna.">Intraflagellar transport motors in cilia: moving along the cell's antenna.</a> J Cell Biol. 180 (1), 23-29 (2008).</li><li>Rosenbaum, J. L., Witman, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraflagellar+transport.">Intraflagellar transport.</a> Nat Rev Mol Cell Biol. 3 (11), 813-825 (2002).</li><li>Ishikawa, H., Marshall, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliogenesis:+building+the+cell's+antenna.">Ciliogenesis: building the cell's antenna.</a> Nat Rev Mol Cell Biol. 12 (4), 222-234 (2011).</li><li>Niwa, 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=Autoinhibition+of+a+Neuronal+Kinesin+UNC-104/KIF1A+Regulates+the+Size+and+Density+of+Synapses.">Autoinhibition of a Neuronal Kinesin UNC-104/KIF1A Regulates the Size and Density of Synapses.</a> Cell Rep. 16 (8), 2129-2141 (2016).</li><li>Snow, J. J., 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=Two+anterograde+intraflagellar+transport+motors+cooperate+to+build+sensory+cilia+on+C.+elegans+neurons.">Two anterograde intraflagellar transport motors cooperate to build sensory cilia on C. elegans neurons.</a> Nat Cell Biol. 6 (11), 1109-1113 (2004).</li><li>Ou, G., Blacque, O. E., Snow, J. J., Leroux, M. R., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coordination+of+intraflagellar+transport+motors.">Functional coordination of intraflagellar transport motors.</a> Nature. 436 (7050), 583-587 (2005).</li><li>Zhou, R., Niwa, S., Homma, N., Takei, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIF26A+is+an+unconventional+kinesin+and+regulates+GDNF-Ret+signaling+in+enteric+neuronal+development.">KIF26A is an unconventional kinesin and regulates GDNF-Ret signaling in enteric neuronal development.</a> Cell. 139 (4), 802-813 (2009).</li><li>Zhao, C., 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=Charcot-Marie-Tooth+disease+type+2A+caused+by+mutation+in+a+microtubule+motor+KIF1Bbeta.">Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta.</a> Cell. 105 (5), 587-597 (2001).</li><li>Niwa, S., Tanaka, Y., Hirokawa, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIF1Bbeta-+and+KIF1A-mediated+axonal+transport+of+presynaptic+regulator+Rab3+occurs+in+a+GTP-dependent+manner+through+DENN/MADD.">KIF1Bbeta- and KIF1A-mediated axonal transport of presynaptic regulator Rab3 occurs in a GTP-dependent manner through DENN/MADD.</a> Nat Cell Biol. 10 (11), 1269-1279 (2008).</li><li>Zhou, R., Niwa, S., Guillaud, L., Tong, Y., Hirokawa, N. <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+motor,+KIF13A,+controls+anxiety+by+transporting+the+serotonin+type+1A+receptor.">A molecular motor, KIF13A, controls anxiety by transporting the serotonin type 1A receptor.</a> Cell Rep. 3 (2), 509-519 (2013).</li><li>Klassen, M. P., 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=An+Arf-like+small+G+protein,+ARL-8,+promotes+the+axonal+transport+of+presynaptic+cargoes+by+suppressing+vesicle+aggregation.">An Arf-like small G protein, ARL-8, promotes the axonal transport of presynaptic cargoes by suppressing vesicle aggregation.</a> Neuron. 66 (5), 710-723 (2010).</li><li>Mondal, S., Ahlawat, S., Koushika, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+microfluidic+devices+for+in+vivo+imaging+of+C.+elegans,+Drosophila+and+zebrafish.">Simple microfluidic devices for in vivo imaging of C. elegans, Drosophila and zebrafish.</a> J Vis Exp. (67), (2012).</li><li>Chai, Y., 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=Live+imaging+of+cellular+dynamics+during+Caenorhabditis+elegans+postembryonic+development.">Live imaging of cellular dynamics during Caenorhabditis elegans postembryonic development.</a> Nat Protoc. 7 (12), 2090-2102 (2012).</li><li>Kim, E., Sun, L., Gabel, C. V., Fang-Yen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+imaging+of+Caenorhabditis+elegans+using+nanoparticle-mediated+immobilization.">Long-term imaging of Caenorhabditis elegans using nanoparticle-mediated immobilization.</a> PLoS One. 8 (1), 53419 (2013).</li><li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+transgenic+C.+elegans+using+microinjection.">Generation of stable transgenic C. elegans using microinjection.</a> J Vis Exp. (18), (2008).</li><li>Collet, J., Spike, C. A., Lundquist, E. A., Shaw, J. E., Herman, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+osm-6,+a+gene+that+affects+sensory+cilium+structure+and+sensory+neuron+function+in+Caenorhabditis+elegans.">Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.</a> Genetics. 148 (1), 187-200 (1998).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Niwa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nephronophthisis-related+gene+ift-139+is+required+for+ciliogenesis+in+Caenorhabditis+elegans.">The nephronophthisis-related gene ift-139 is required for ciliogenesis in Caenorhabditis elegans.</a> Sci Rep. 6, 31544 (2016).</li><li>Maeder, C. I., San-Miguel, A., Wu, E. Y., Lu, H., Shen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+neuron-wide+analysis+of+synaptic+vesicle+precursor+trafficking.">In vivo neuron-wide analysis of synaptic vesicle precursor trafficking.</a> Traffic. 15 (3), 273-291 (2014).</li><li>Wu, Y. E., Huo, L., Maeder, C. I., Feng, W., Shen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+balance+between+capture+and+dissociation+of+presynaptic+proteins+controls+the+spatial+distribution+of+synapses.">The balance between capture and dissociation of presynaptic proteins controls the spatial distribution of synapses.</a> Neuron. 78 (6), 994-1011 (2013).</li><li>Dwyer, N. D., Adler, C. E., Crump, J. G., L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+dendritic+transport+and+the+AP-1+mu1+clathrin+adaptor+UNC-101+localize+odorant+receptors+to+olfactory+cilia.">Polarized dendritic transport and the AP-1 mu1 clathrin adaptor UNC-101 localize odorant receptors to olfactory cilia.</a> Neuron. 31 (2), 277-287 (2001).</li><li>Fang-Yen, C., Gabel, C. V., Samuel, A. D., Bargmann, C. I., Avery, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microsurgery+in+Caenorhabditis+elegans.">Laser microsurgery in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 177-206 (2012).</li><li>Kumar, J., 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+Caenorhabditis+elegans+Kinesin-3+motor+UNC-104/KIF1A+is+degraded+upon+loss+of+specific+binding+to+cargo.">The Caenorhabditis elegans Kinesin-3 motor UNC-104/KIF1A is degraded upon loss of specific binding to cargo.</a> PLoS Genet. 6 (11), 1001200 (2010).</li><li>Zheng, Q., 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+vesicle+protein+SAM-4+regulates+the+processivity+of+synaptic+vesicle+transport.">The vesicle protein SAM-4 regulates the processivity of synaptic vesicle transport.</a> PLoS Genet. 10 (10), 1004644 (2014).</li><li>Blacque, O. 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=The+WD+repeat-containing+protein+IFTA-1+is+required+for+retrograde+intraflagellar+transport.">The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport.</a> Mol Biol Cell. 17 (12), 5053-5062 (2006).</li><li>Evans, J. 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=Functional+modulation+of+IFT+kinesins+extends+the+sensory+repertoire+of+ciliated+neurons+in+Caenorhabditis+elegans.">Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans.</a> J Cell Biol. 172 (5), 663-669 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat Methods. 2 (12), 905-909 (2005).</li><li>Bindels, D. 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=mScarlet:+a+bright+monomeric+red+fluorescent+protein+for+cellular+imaging.">mScarlet: a bright monomeric red fluorescent protein for cellular imaging.</a> Nat Methods. 14 (1), 53-56 (2017).</li><li>Prevo, B., Mangeol, P., Oswald, F., Scholey, J. M., Peterman, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+differentiation+of+cooperating+kinesin-2+motors+orchestrates+cargo+import+and+transport+in+C.+elegans+cilia.">Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia.</a> Nat Cell Biol. 17 (12), 1536-1545 (2015).</li><li>Robison, P., 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=Detyrosinated+microtubules+buckle+and+bear+load+in+contracting+cardiomyocytes.">Detyrosinated microtubules buckle and bear load in contracting cardiomyocytes.</a> Science. 352 (6284), 0659 (2016).</li><li>Hayashi, S., Okada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+superresolution+fluorescence+imaging+with+spinning+disk+confocal+microscope+optics.">Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics.</a> Mol Biol Cell. 26 (9), 1743-1751 (2015).</li></ol>

作者没有什么要透露的。

关于现有方法的限制 本文所描述的方法进行了优化, 以观察快速事件, 如轴突运输和 IFT。因此, 固定化比较长的潜伏期更有优先权。虽然我们已经能够观察贩运活动至少20分钟没有明显的摄动, 这种方法可能并不总是适合观察慢事件需要更长的观察, 如轴突伸长和细胞迁移。对于较长的观测, 需要通过减少琼脂糖的百分比 (即, 增加水以避免干燥和减少可能造成损坏的压力) 来优?...

活细胞成像是分析细胞内转运的重要工具。在神经元细胞生物学中, 用活细胞成像分析轴突转运是了解神经元功能和形态发生1的必要条件。轴突运输缺陷的基础上几个神经退行性疾病2。驱动超家族蛋白和蛋白进行轴突转运 anterogradely 和逆行, 分别为1,2。
纤毛是另一个细胞室, 其中微管网络和贩卖机械是高度发达的3。蛋白质合成机械没有在纤毛中定位, 这意味着纤毛蛋白必须从细胞质输送到纤毛的尖端。纤毛特异性驱动和蛋白, 分别称为 kinesin-2 和细胞质 dynein-2, 运输的成分纤毛4, 在一个现象称为 intraflagellar 运输 (IFT)5。IFT 的损害导致一系列疾病称为 ciliopathies6。因此, 需要对 IFT 机制进行活体细胞成像分析, 以了解纤毛形成的基本机制和发病机理。
线虫线虫(线虫) 是研究轴突传输和 IFT7、8、9的好模型。为了观察 IFT,衣藻已被广泛用作模型生物体56。由于衣藻是一个单细胞生物体, IFT 与衰老、神经元功能和行为的关系将难以分析。此外, 诸如 CRISPR/Cas9 等基本遗传技术尚未应用到衣藻。在高模型生物体中, 如小鼠和果蝇, 轴突转运和 IFT 的中断经常导致致命的表型, 因为轴突转运和 IFT 对动物的形态发生和稳态至关重要10, 11。在小鼠的情况下, 细胞培养和转染一般需要观察轴突运输和 IFT, 这需要很多技能和大量的时间12,13。此外, 许多重要的生理环境可能会失去在培养细胞和细胞系。因为神经系统对于蠕虫的生存来说不是必需的, 所以轴突运输或 IFT 被破坏的线虫突变体通常不是致命的7、9、14。轴突运输和 IFT 可以直接观察在体内没有解剖, 因为C. 线虫是透明的, 因此很容易观察 GFP 标记标记。
有几种协议可以固定C. 线虫, 例如使用微流体设备15、带有麻醉的琼脂糖垫16或微17。麻醉的纳入可能抑制神经元的贩运事件15。微流控装置方法的一个明显的缺点是, 准备一台流控装置并不总是容易的。相反, 用琼脂糖垫和微固定是一种方便和简单的方法来执行时间推移成像在C. 线虫。在这里, 我描述这个基本的协议, 以固定的线虫和可视化轴突运输和 IFT在体内在线虫.与其他方法相比, 这里所描述的方法不需要特殊设备, 而且更便宜、更容易执行。

<table><tbody><tr><td>Slideglass (76&amp;nbsp; x 26 mm)</td><td>Matsunami</td><td>S1111</td><td></td></tr><tr><td>Coverglass (22&amp;nbsp; x 40 mm)</td><td>Matsunami</td><td>C024401</td><td></td></tr><tr><td>Agarose</td><td>Wako</td><td>318-01195</td><td></td></tr><tr><td>Polystylene microbeads 0.1 micron</td><td>Polysciences</td><td>#00876</td><td></td></tr><tr><td>Heat block</td><td>TAITEC</td><td>0063288-000</td><td>CTU-mini</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX-71</td><td>widefield microscope</td></tr><tr><td>Spinning disk Scanner</td><td>Yokogawa</td><td>CSU-X1</td><td>spinning disc confocal scanner&amp;nbsp;</td></tr><tr><td>Digital CCD camera</td><td>Hamamatsu Photonics</td><td>C10600-10B</td><td>ORCA-R2 degital CCD camera</td></tr><tr><td>Objective lens (x100, NA1.4)</td><td>Olympus</td><td>UPLSAPO 100XO</td><td></td></tr><tr><td>Pasteur pipette (5 inch)</td><td>IWAKI</td><td>IK-PAS-5P</td><td></td></tr><tr><td>Glass tube (1.5 cm diameter x 10.5 cm)</td><td>IWAKI</td><td>9820TST15-105NP</td><td></td></tr><tr><td>TV9211: &lt;em&gt;wyIs251&lt;/em&gt;</td><td>Laboratory of Kang Shen</td><td>N/A</td><td></td></tr><tr><td>OTL11: &lt;em&gt;mnIs17&lt;/em&gt;</td><td>Ref. 27, 29</td><td>N/A</td><td>SP2101 was backcrossed with wild type for 6 times</td></tr><tr><td>Stereo microscope</td><td>Carl Zeiss</td><td>435064-9000-000</td><td>STEMI 508&amp;nbsp;</td></tr><tr><td>Mirror transillumination unit</td><td>Carl Zeiss</td><td>435425-9010-000</td><td></td></tr><tr><td>platinum wire (0.2 mm)</td><td>Nilaco Corporation</td><td>m78483501</td><td></td></tr><tr><td>60 mm plastic dish</td><td>Falcon</td><td>#351007</td><td></td></tr><tr><td>Fiji</td><td>N/A</td><td>N/A</td><td>https://fiji.sc/</td></tr><tr><td>nematode growth medium (NGM)&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td>1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4&amp;nbsp;</td></tr></tbody></table>

immobilization of caenorhabditis elegans to analyze intracellular transport in neurons

轴突运输和 intraflagellar 转运 (IFT) 是轴索和纤毛形态发生和功能的关键。驱动超家族蛋白和蛋白是分别调节顺和逆行转运的分子马达。这些马达使用微管网络作为路轨。线虫线虫(线虫) 是研究轴突转运和 IFT的一个强有力的模型有机体。在这里, 我描述了一个协议, 以观察轴突运输和 IFT 在生活中的C. 线虫。运输货物可以通过使用荧光蛋白 (如绿色荧光蛋白 (GFP)) 标记货物蛋白质来可视化。C. 线虫是透明的, 而 GFP 标记的货物蛋白可以在特定细胞的细胞特异促进剂下表达。活蠕虫可以通过微10% 琼脂糖凝胶固定, 而不会杀死或麻醉蠕虫。在这种情况下, 货物的运动可以直接观察到的轴突和纤毛的生活线虫没有解剖。这种方法可用于观察任何细胞的任何货物分子通过修改目标蛋白和/或他们所表达的细胞。大多数基本的蛋白质, 如分子马达和适配器蛋白, 参与轴突运输和 IFT 是保存在C. 线虫。与其他模型生物体相比, 突变体可以在C. 线虫中更容易获得和维护。将此方法与各种C. 线虫突变体结合, 可以阐明轴突转运和 IFT 的分子机制。

DA9 神经元轴突转运 使用wyIs251线, 顺和逆行轴突运输 GFP::RAB-3 可以同时记录在 DA9 运动神经元。DA9 神经元近端轴突的顺和逆行转运的平均速度分别为1.8 和2.6 微米/秒,22。移动的泡的数量是大约0.03 和0.018 每μ m 轴突每 s。因此, 三十年代观察10μ m DA9 轴突使用100x 透镜, 你能发现大约9和5移动的泡在轴突 (图 4和<strong...

Watch this Scientific Journal Video about Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons at JoVE.com

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

线虫线虫(线虫) 是研究轴突和细胞内转运的良好模型。在这里, 我描述了一个协议的在体内记录和分析轴突和 intraflagellar 运输在C. 线虫。

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

immobilization-caenorhabditis-elegans-to-analyze-intracellular

Research

JoVE Journal

Developmental Biology

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

8.0K 次观看.  Tohoku University. 线虫线虫(线虫) 是研究轴突和细胞内转运的良好模型。在这里, 我描述了一个协议的在体内记录和分析轴突和 intraflagellar 运输在C. 线虫。

视频: Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

科研

生物化学

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

神经科学

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...

发育生物学

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

生物工程

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the neurotransmitter glutamate, and the excessive stimulation of its postsynaptic receptors. However, information on the subsequent molecular events that culminate in the distinct neuronal swelling morphology of this type of neurodegeneration is lacking. Other aspects, such as changes in specific subcellular compartments, or the basis for the differential cellular vulnerability of distinct neuronal subtypes, remain under-explored. Furthermore, a range of factors that come into play in studies that use in vitro or ex vivo preparations might modify and distort the natural progression of this form of neurodegeneration. It is therefore important to study excitotoxic necrosis in live animals by monitoring the effects of interventions that regulate the extent of neuronal necrosis in the genetically amenable and transparent&#160;model system of the nematode Caenorhabditis elegans. This protocol describes methods of studying excitotoxic necrosis in C. elegans neurons, combining optical, genetic, and molecular analysis. To induce excitotoxic conditions in C. elegans, a&#160;knockout of a glutamate transporter gene (glt-3) is combined with a neuronal sensitizing genetic background (nuls5 [Pglr-1::G&#945;S(Q227L)]) to produce glutamate receptor hyperstimulation and neurodegeneration. Nomarski differential interference contrast (DIC), fluorescent, and confocal microscopy in live animals are methods used to quantify neurodegeneration, follow subcellular localization of fluorescently labeled proteins, and quantify mitochondrial morphology in the degenerating neurons. Neuronal Fluorescence Activated Cell Sorting (FACS) is used to distinctly sort at-risk neurons for cell-type specific transcriptomic analysis of neurodegeneration. A combination of live imaging and FACS methods as well as the benefits of the C. elegans model organism allow researchers to leverage this system to obtain reproducible data with a large sample size. Insights from these assays could translate to novel targets for therapeutic intervention in neurodegenerative diseases.

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

In a C. elegans excitotoxicity model, this protocol employs in vivo imaging to analyze the regulation of necrotic neurodegeneration, the effect of genes encoding candidate mediators, and involvement of mitochondria. Cell dissociation and sorting is used to specifically obtain at-risk neurons for cell-specific transcriptomic analysis of neurodegeneration and neuroprotection mechanisms.

Excitotoxic necrosis is a leading form of neurodegeneration. This process of regulated necrosis is triggered by the synaptic accumulation of the ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Subcellular Imaging of Neuronal Calcium Handling In Vivo

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,&#160;where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

Subcellular Imaging of Neuronal Calcium Handling In Vivo

The current methods describe a non-ratiometric approach for high-resolution, sub-compartmental calcium imaging in vivo in Caenorhabditis elegans using readily available genetically encoded calcium indicators.

体内神经元钙处理的亚细胞成像

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a ...

建造和操作冷却阶段以集体固定秀丽隐杆线虫

Assembly and Operation of a Cooling Stage to Immobilize C. elegans on Their Culture Plates

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), but require strong animal immobilization to prevent motion blur in the images. Unfortunately, most current immobilization techniques require substantial manual effort, rendering high-resolution imaging low-throughput. Immobilization of C. elegans is greatly simplified by using a cooling approach that can easily immobilize entire populations directly on their cultivation plates. The cooling stage can establish and maintain a wide range of temperatures with a uniform distribution on the cultivation plate. In this article, the whole process of building the cooling stage is documented. The aim is that a typical researcher can build an operational cooling stage in their laboratory following this protocol without difficulty. Utilization of the cooling stage following three protocols is shown, and each protocol has advantages for different experiments. Also shown is an example cooling profile of the stage as it approaches its final temperature and some helpful tips in using cooling immobilization.

作者聚焦： 组装和操作冷却台以将秀丽隐杆线虫固定在培养板上  

This paper describes protocols for constructing and operating a cooling stage to immobilize C. elegans on their original cultivation plates en masse.

组装和操作冷却阶段，以固定 秀丽隐杆线虫 在其培养板上

High-resolution in vivo microscopy approaches can reveal subtle information and fine details inside the model animal Caenorhabditis elegans (C. elegans), ...

固定化的线虫线虫分析神经元内细胞的转运

摘要

探索更多视频

此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

琼脂糖微室的长期钙成像

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

体内神经元钙处理的亚细胞成像

组装和操作冷却阶段，以固定秀丽隐杆线虫在其培养板上

组装和操作冷却阶段，以固定秀丽隐杆线虫在其培养板上

固定化的线虫线虫分析神经元内细胞的转运

摘要

探索更多视频

此视频中的章节

在 Vivo 钙成像中，在 C. elegans身体壁肌肉

C.在体内神经元钙成像技术线虫

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

琼脂糖微室的长期钙成像

用于秀丽隐杆线虫长期生长和成像的简单微流控芯片

Live Animal Imaging and Cell Sorting Methods for Investigating Neurodegeneration in a C. elegans Excitotoxic Necrosis Model

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

体内神经元钙处理的亚细胞成像

组装和操作冷却阶段，以固定 秀丽隐杆线虫 在其培养板上

组装和操作冷却阶段，以固定 秀丽隐杆线虫 在其培养板上

组装和操作冷却阶段，以固定秀丽隐杆线虫在其培养板上

组装和操作冷却阶段，以固定秀丽隐杆线虫在其培养板上