我们要感谢岳（保罗）胡锦涛，雷切尔SINIT为他们提供技术援助。这项研究是由美国国立卫生研究院资助（PN2 EY016525），并从唐氏综合症的研究与治疗基金会和拉里·L.希尔布鲁姆基金会的资助支持。

手术和动物的程序进行了严格按照美国国立卫生研究院指南的护理和使用实验动物。所有涉及使用动物实验是由加州大学圣地亚哥分校机构动物照顾及使用委员会批准。 1质粒的克隆，单生物素BDNF的表达和纯化（mBtBDNF） 注:建设前期proBDNFavi和BirA的cDNA插入在HEK293FT细胞10 pcDNA3.1载体和共表达。使用Ni-NTA珠根据生产成熟的和生物活性monobiotinylated神经生长因子（mBtNGF）10的先前公布的方法mBtBDNF纯化。 2，制备微流控商会微流体神经元培养装置，能够使流体流动的隔离神经元胞体的轴突。每个解剖前集会权与新涂盖玻片室。用于微流控室在这个协议是商业购买（见材料和​​设备的表）。手洗和重复购买商业腔高达5-6x。 <ol><li>手洗的1％Alconox清洁剂的微流控室。漂洗三次，用Milli-Q水用于各30分钟。再次手洗室70％的乙醇。 </li><li>铺陈室晾干的封口膜的层流罩。辐射灭菌的腔两侧紫外光下20分钟。存放在无菌15cm培养皿在室温下用石蜡膜密封消毒室。 </li><li>将24×40mm的1号盖玻片在玻璃容器中。浸泡过夜旋转器上在35％盐酸盖玻片。在第二天，冲洗盖玻片三次，每次加水30分钟。 </li><li>浸渍每个盖玻片到100％的乙醇和燃烧过本生灯消毒盖玻片一个接一个。储存干盖玻片在无菌培养皿中，并保持在室温下直至涂层。 </li><li>莱OU吨，15厘米的培养皿中的盖玻片。外套每个盖玻片用0.7ml的0.01％的聚-L-赖氨酸（PLL），并培育在通风橱中在室温下进行。 </li><li> 1小时后，冲洗盖玻片三次，用无菌水。干盖玻片真空和地方每个盖玻片成一块2.4英寸的培养皿。 </li><li>组装微流体室中，放置与微沟槽侧的腔室的底部上的PLL涂覆的盖玻片小心不要接触到微槽。轻轻按下室用枪头，以确保室内进行密封。 </li></ol> 3，神经解剖文化和板块上庭<ol><li>放置两个E17-E18大鼠海马（一个脑）解剖在15ml锥形管中含2ml夹层缓冲液（HBSS，无钙无镁，用1％青霉素/链霉素和10mM HEPES。冲洗组织3倍用5ml每次清扫缓冲拆下清扫缓冲尽可能并添加900微升新鲜二ssection缓冲区。 </li><li>消化组织，加入100μl的10X胰蛋白酶（2.5％）进夹层缓冲，使1个工作浓度。放置锥形管在37℃水浴。消化后的10分钟，加入100μl的10毫克/毫升的DNase I至约1毫克/毫升的最终浓度。 </li><li>用火抛光的巴斯德玻璃吸管，轻轻地上下吹打5到10倍磨碎的组织。研磨后右，淬火用2ml平板培养基（神经基础有10％FBS，2mM的Glutamax的，2％B27）的胰蛋白酶。 </li><li>留在引擎盖样品5分钟，使组织中的任何碎片落户到了谷底。小心地取出2毫升上清液倒入干净无菌的15毫升锥形管中，离心分离机，在200×g离心5分钟以沉淀细胞。悬浮颗粒在50微升电镀媒体</li><li>计数细胞与血球。负载15-20微升细胞悬浮液（〜40,000个细胞）到微流体室中的一个隔室。放置室在培养箱中培养10分钟，以使细胞附着在盖玻片上。 10分钟后，加入更多的镀介质来填充该腔室的两个室。 </li><li>对解剖的第二天，完全（有2mM Glutamax的，2％B27的Neurobasal）在两个细胞体和轴突隔室更换电镀媒体与维护媒体。从海马神经元的轴突开始穿越微槽3天，达到5-7天之间的轴突舱。在这段时间内，用新鲜维持培养基，每24〜48小时，更换一半的培养介质。 </li></ol> QD-BDNF的4轴突运输<ol><li>之前住QD-BDNF的轴突运输的成像，从微流体室中的两个细胞体和轴突隔室耗尽BDNF通过彻底冲洗两个室用BDNF无，无血清的Neurobasal介质，每30分钟2小时。 </li><li>在BDNF的枯竭，准备QD-BDNF的结合物。搭配50纳米的单生物素化的BDNF二聚体用50nM的Neurobasal介质QD655-链霉抗生物共轭物和在冰上孵育60分钟。 </li><li>在轴突舱取出介质，加入300μlQD-BDNF 0.25纳米的终浓度为4小时，在37℃。为了尽量减少QD-BDNF的漫射到细胞体隔室，这是很重要的，以始终保持介质中的细胞体隔室更高的水平比在轴突隔室。前实时成像孵育后洗去未结合的QD-BDNF。 </li><li>使用倒置显微镜配有100X油物镜进行QD-BDNF运输的实时成像。预热的范围和附加到它的恒定温度（37℃）和CO 2（5％）的环境室中。使用一组德克萨斯红的激发/发射的多维数据集可视化的QD655信号。 </li><li>获取和捕捉中间的轴突内的延时图像1帧/秒的速度，总共2分钟使用CCD摄像头。使用细微沟槽，无轴突塔吨有没有量子点，因此没有信号的渗透控制。 </li><li>分析使用任何图像分析软件或NIH ImageJ的BDNF的运输。 </li></ol>

<ol>
	<li>Wu, 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=A+functional+dynein-microtubule+network+is+required+for+NGF+signaling+through+the+Rap1/MAPK+pathway.">A functional dynein-microtubule network is required for NGF signaling through the Rap1/MAPK pathway.</a> Traffic. 8, 1503-1520 (2007).</li><li>Wortzel, I., Seger, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ERK+Cascade:+Distinct+Functions+within+Various+Subcellular+Organelles.">The ERK Cascade: Distinct Functions within Various Subcellular Organelles.</a> Genes &amp; cancer. 2, 195-209 (2011).</li><li>Huang, E. J., Reichardt, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trk+receptors:+roles+in+neuronal+signal+transduction.">Trk receptors: roles in neuronal signal transduction.</a> Annual review of biochemistry. 72, 609-642 (2003).</li><li>Nonomura, 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=Signaling+pathways+and+survival+effects+of+BDNF+and+NT-3+on+cultured+cerebellar+granule+cells.">Signaling pathways and survival effects of BDNF and NT-3 on cultured cerebellar granule cells.</a> Brain research. Developmental brain research. 97, 42-50 (1996).</li><li>Weissmiller, A. M., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+in+using+neurotrophic+factors+to+treat+neurodegenerative+disorders.">Current advances in using neurotrophic factors to treat neurodegenerative disorders.</a> Translational neurodegeneration. 1, 14 (2012).</li><li>Zhang, 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=Defective+axonal+transport+of+Rab7+GTPase+results+in+dysregulated+trophic+signaling.">Defective axonal transport of Rab7 GTPase results in dysregulated trophic signaling.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 33, 7451-7462 (2013).</li><li>Taylor, A. 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=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature methods. 2, 599-605 (2005).</li><li>Cui, B., 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=One+at+a+time,+live+tracking+of+NGF+axonal+transport+using+quantum+dots.">One at a time, live tracking of NGF axonal transport using quantum dots.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 13666-13671 (2007).</li><li>Xie, W., Zhang, K., Cui, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+and+axonal+transport+of+quantum+dot+labeled+BDNF.">Functional characterization and axonal transport of quantum dot labeled BDNF.</a> Integrative biology : quantitative biosciences from nano to macro. 4, 953-960 (2012).</li><li>Sung, K., Maloney, M. T., Yang, J., Wu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+producing+mono-biotinylated,+biologically+active+neurotrophic+factors:+an+essential+reagent+for+single+molecule+study+of+axonal+transport.">A novel method for producing mono-biotinylated, biologically active neurotrophic factors: an essential reagent for single molecule study of axonal transport.</a> Journal of neuroscience methods. 200, 121-128 (2011).</li><li>Tani, 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=Trafficking+of+a+ligand-receptor+complex+on+the+growth+cones+as+an+essential+step+for+the+uptake+of+nerve+growth+factor+at+the+distal+end+of+the+axon:+a+single-molecule+analysis.">Trafficking of a ligand-receptor complex on the growth cones as an essential step for the uptake of nerve growth factor at the distal end of the axon: a single-molecule analysis.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 25, 2181-2191 (2005).</li><li>Bronfman, F. C., Tcherpakov, M., Jovin, T. M., Fainzilber, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-induced+internalization+of+the+p75+neurotrophin+receptor:+a+slow+route+to+the+signaling+endosome.">Ligand-induced internalization of the p75 neurotrophin receptor: a slow route to the signaling endosome.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 23, 3209-3220 (2003).</li><li>Kruttgen, A., Heymach, J. V., Kahle, P. J., Shooter, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+nerve+growth+factor+carboxyl+terminus+in+receptor+binding+and+conformational+stability.">The role of the nerve growth factor carboxyl terminus in receptor binding and conformational stability.</a> The Journal of biological chemistry. 272, 29222-29228 (1997).</li><li>Zuccato, C., Cattaneo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+in+neurodegenerative+diseases.">Brain-derived neurotrophic factor in neurodegenerative diseases.</a> Nature reviews. Neurology. 5, 311-322 (2009).</li><li>Gharami, K., Xie, Y., An, J. J., Tonegawa, S., Xu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain-derived+neurotrophic+factor+over-expression+in+the+forebrain+ameliorates+Huntington's+disease+phenotypes+in+mice.">Brain-derived neurotrophic factor over-expression in the forebrain ameliorates Huntington's disease phenotypes in mice.</a> Journal of neurochemistry. 105, 369-379 (2008).</li><li>Gauthier, L. 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=Huntingtin+controls+neurotrophic+support+and+survival+of+neurons+by+enhancing+BDNF+vesicular+transport+along+microtubules.">Huntingtin controls neurotrophic support and survival of neurons by enhancing BDNF vesicular transport along microtubules.</a> Cell. 118, 127-138 (2004).</li><li>Her, L. S., Goldstein, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+of+striatal+neurons+to+axonal+transport+defects+induced+by+mutant+huntingtin.">Enhanced sensitivity of striatal neurons to axonal transport defects induced by mutant huntingtin.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 28, 13662-13672 (2008).</li><li>Rong, 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=Regulation+of+intracellular+trafficking+of+huntingtin-associated+protein-1+is+critical+for+TrkA+protein+levels+and+neurite+outgrowth.">Regulation of intracellular trafficking of huntingtin-associated protein-1 is critical for TrkA protein levels and neurite outgrowth.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 26, 6019-6030 (2006).</li></ol>

没有利益冲突的声明。

在这项研究中，我们报道了一种新技术的发展，以产生具有生物活性，monobiotinylated BDNF（mBtBDNF），可以用来跟踪BDNF的轴突运输。通过缀合的蛋白质，以量子点的链霉亲和使用的微流体腔室，该方法允许一个检测BDNF在与单个分子的灵敏度，在实时的和具有空间分辨率和时间分辨率原代神经元的轴突运输。这里所使用的工具提供了由研究介导的BDNF / TrkB的信号内吞体的健康和疾病的神经元轴突运输?...

神经元是高度分化的细胞，其长，通常高度阐述的过程是基本建立和保持神经回路的结构和功能。轴突起着运载的货物和从突触至关重要的作用。蛋白质和细胞器的细胞胞体合成需要通过轴突运输到达突触前末梢，支持神经元功能。相应地，接收的信号在远端轴突需要被转并输送到体细胞。这些方法可用于神经元的存活，分化和维持所必需的。在某些神经元的轴突运输必须通过的距离超过1000倍的细胞体的直径进行，有可能容易地预想到，即使是很小的缺陷可显着影响神经元和电路功能。 脑源性神经营养因子（BDNF），生长因子的神经营养因子家族中的一员，是目前在马纽约州的大脑区域，包括海马，大脑皮层和基底前脑。 BDNF起着认知和记忆的形成所配套的存活，分化，并参与认知神经回路的功能至关重要的作用。 BDNF结合到其受体的酪氨酸激酶受体TrkB，在轴突终端那里它激活的TrkB介导的信号传导途径，包括有丝分裂原激活的蛋白激酶/细胞外信号调节蛋白激酶（MAPK / ERK），磷脂酰肌醇3激酶（PI3K）和磷脂酶C-γ（PLCγ）。在参与这些信号传导途径的蛋白被包装到内吞囊泡结构，以形成BDNF / TrkB的信令内体1-6被再逆行转运到神经元胞体。 微流体培养室是研究在正常条件下，以及在损伤和疾病7,8的设定轴突生物学一个非常有用的平台。通过隔离轴突从细胞体，该装置允许一个具体研究运输中的轴突8-10。在与在该研究中使用450微米的微沟槽屏障基于PDMS的微流体平台进行商业购买（参见材料表）。为了研究BDNF的运输，我们开发了一种新的技术，生产monobiotinylated BDNF（mBtBDNF）。我们把生物素受体肽，美联社（又称重组蛋白质）的优势。它是一个包含一个赖氨酸残基，可以由大肠杆菌酶的生物素连接酶，BirA的具体连接到生物素15的氨基酸序列。我们融合的重组蛋白质的小鼠预先proBDNF基因的C末端，通过PCR（ 图1A）。该构建体被克隆到哺乳动物表达载体pcDNA3.1 myc基因-His载体。我们还克隆的细菌BirA的DNA导入的pcDNA3.1 myc基因-His载体。两个质粒瞬时共转染到HEK293FT细胞表达两种蛋白。 BirA的催化毕结扎在具体的赖氨酸驻留在重组蛋白质中的BDNF的在1的C末端:1的比例以产生monobiotinylated BDNF单体。生物素化的，成熟的BDNF与〜18kDa的分子质量，回收和使用的Ni-树脂（ 图1C）的介质纯化。 BDNF的生物素化是完全的，作为判断无法通过免疫印迹法（ 图1D），以检测未修饰BDNF。链霉亲和共轭量子点，量子点655，分别用来标明mBtBDNF使QD-BDNF。的重组蛋白质的存在下不与BDNF的活性干扰作为mBtBDNF能够激活磷酸化的TrkB（ 图1E）和刺激神经 ​​突向外生长（ 图1F），以重组人BDNF（rhBDNF）的程度。免疫组化显示，QD-BDNF共定位与TrkB的海马神经轴突，表明QD-BDNF的生物活性（ 图1G）。研究了BDNF的运输，QD-BDNF加入到远端轴突隔室含E18大鼠海马神经元（ 图2A）微流控文化。在轴突QD-BDNF逆行运输被抓获的红色荧光标记（ 支持视频S1，S2）的实时现场图像。通过分析所产生的kymograph，QD-BDNF的观察可以在约1.06微米/秒（ 图3A）的移动速度逆向转移。 GFP或mCherry-标记的BDNF已被用于跟踪BDNF的神经轴突的运动。主要的缺点是，他们不是为单个分子的研究不够明亮。另外，无论是顺行性与逆行BDNF运动的存在使得难以评估的逆向转移BDNF是否是一种神经营养蛋白/受体复合体。 在这段视频中，我们展示了用于检查QD-BDNF在利用原代神经元的微流控室现场贩卖的技术。该ultrabrightness和量子点麦出色的耐光性ES能够进行BDNF传输的长期跟踪。这些技术可被利用来提高AD中，HD和其他神经变性疾病的轴突功能的研究。

<table><tbody><tr><td>Platinum pfx DNA polymerase&amp;nbsp;</td><td>Invitrogen</td><td>11708021</td><td></td></tr><tr><td>EcoRI&amp;nbsp;</td><td>Fermentas</td><td>FD0274</td><td></td></tr><tr><td>BamHI&amp;nbsp;</td><td>Fermentas</td><td>FD0054</td><td></td></tr><tr><td>HEK293FT cells</td><td>Invitrogen</td><td>R70007</td><td></td></tr><tr><td>DMEM-high glucose media</td><td>Mediatech</td><td>10-013-CV</td><td></td></tr><tr><td>D-biotin&amp;nbsp;</td><td>Sigma</td><td>B4639</td><td></td></tr><tr><td>TurboFect&amp;nbsp;</td><td>Fermentas</td><td>R0531</td><td></td></tr><tr><td>PMSF&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A6279</td><td></td></tr><tr><td>Ni-NTA resins</td><td>Qiagen</td><td>30250</td><td></td></tr><tr><td>Protease inhibitors cocktail</td><td>Sigma</td><td>&amp;nbsp;S8820</td><td></td></tr><tr><td>Silver staining kit&amp;nbsp;</td><td>G-Biosciences</td><td>786-30</td><td></td></tr><tr><td>Human recombinant BDNF</td><td>Genentech</td><td></td><td></td></tr><tr><td>Microfluidic chambers</td><td>Xona</td><td>SND450</td><td></td></tr><tr><td>24 x 40 mm No. 1 glass coverslips&amp;nbsp;</td><td>VWR</td><td>48393-060</td><td></td></tr><tr><td>Poly-L-Lysine&amp;nbsp;</td><td>Cultrex</td><td>3438-100-01</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14185-052</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090-046</td><td></td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>GlutaMax&amp;nbsp;</td><td>Invitrogen</td><td>35050-061</td><td></td></tr><tr><td>B27&amp;nbsp;&amp;nbsp;</td><td>Gibco</td><td>17504-044</td><td></td></tr><tr><td>QD655-streptavidin conjugates</td><td>Invitrogen</td><td>&amp;nbsp;Q10121MP</td><td></td></tr><tr><td>anti-Avi tag antibody</td><td>GenScript</td><td>A00674</td><td></td></tr><tr><td>streptavidin-agarose beads&amp;nbsp;</td><td>Life Technology</td><td>&amp;nbsp;SA100-04</td><td></td></tr><tr><td>Trichloroacetic acid</td><td>Sigma</td><td>T6399</td><td></td></tr><tr><td>HRP-streptavidin&amp;nbsp;</td><td>Thermo Scientific</td><td>N100</td><td></td></tr><tr><td>anti-pTrkB antibody</td><td>a generous gift from Dr M. Chao of NYU</td><td></td><td></td></tr><tr><td>anti-TrkB antibody</td><td>BD Science</td><td>610101</td><td></td></tr></tbody></table>

real-time imaging of axonal transport of quantum dot-labeled bdnf in primary neurons

BDNF在神经元存活，分化和功能的若干方面具有重要作用。结构和功能的缺陷在轴突日益被看作神经变性疾病，包括阿尔茨海默氏病（AD）和亨廷顿病（HD）的早期特征。尚不清楚是由轴索损伤诱导机制（S）。我们报道了一种新技术的发展，以产生具有生物活性，monobiotinylated BDNF（mBtBDNF），可以用来跟踪BDNF的轴突运输。量子点标记的BDNF（QD-BDNF）是由共轭量子点655到mBtBDNF制备。一种微流体装置被用于分离从神经元胞体的轴突。除了QD-BDNF的到轴突舱允许的BDNF运输轴突的实时成像。我们证明了QD-BDNF的移动基本上是完全逆行，除了极少数停顿，约为1.06微米/秒的移动速度。此系统可用于研究我的破坏轴突功能在AD或HD，以及其他退行性疾病chanisms。

生产具有生物活性的单 - 生物素化的BDNF的纯化 BDNF的稠合有一个重组蛋白质序列（GGGLNDIFEAQKIEWHE）的表达载体，根据以前发表的协议10创建。全长融合蛋白的分子质量预测为〜32kDa的（ <a href="http://ca.expasy.org/tools/pi_tool.html" target="_blank">http://ca.expasy.org/tools/pi_tool.html</a> ）Monobiotinylated成熟的BDNF为18 kDa的（ 图1A）的预测分子量制作在HEK293FT细胞和纯化的条件培?...

Watch this Scientific Journal Video about Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons at JoVE.com

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF，神经营养因子，对轴突运输是几个神经元群体的生存和功能是至关重要的。一些退行性疾病的标志是轴索结构和功能的破坏。我们展示了用于检查QD-BDNF在使用初级神经元微流控室现场贩卖的技术。

初级量子神经元轴突运输的实时成像点标记的BDNF

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

real-time-imaging-axonal-transport-quantum-dot-labeled-bdnf-primary

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

15.0K 次观看.  University of California, San Diego.  BDNF，神经营养因子，对轴突运输是几个神经元群体的生存和功能是至关重要的。一些退行性疾病的标志是轴索结构和功能的破坏。我们展示了用于检查QD-BDNF在使用初级神经元微流控室现场贩卖的技术。 

视频: Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

生物学

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

用于神经元细胞贩运研究的管子中纯化和直接单生物素重组BDNF的改进协议

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

使用稀疏腺相关病毒标记遗传靶向视网膜细胞群的神经元树化延时成像

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

扩展轴突转运体内成像工具包

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

<meta charset="utf8"></head><body>通过活细胞成像可视化原代海马神经元中的蛋白质

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>作者聚焦：了解病理蛋白对神经退行性疾病轴突转运的影响

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.