这项工作是从美国国立卫生研究院（赠款R21的NS45037和以R01 ES014901）的资金支持。

1。培养基的制备（C2的介质） <ol><li>中列出的顺序，添加以下，无菌的，一次性的500毫升三角瓶： <table border="1"><tbody><tr><td> 量 </td><td> 元件 </td><td> 终浓度 </td></tr><tr><td>第190笔资料毫升</td><td> DMEM培养基（低糖） </td><td> N / A </td></tr><tr><td> 10毫升</td><td>脂肪酸免费BSA（DMEM培养液20mg/mL时） </td><td> 50微克/毫升</td></tr><tr><td> 2.8毫升</td><td> L-谷氨酰胺</td><td> 1.4毫米</td></tr><tr><td> 4毫升</td><td>胰岛素/转/硒（100X） </td><td>胰岛素10微克/毫升5.5微克/毫升转38.7纳米硒</td></tr><tr><td> 0.4毫升</td><td>神经生长因子（125微克/毫升） </td><td> 100毫微克/毫升</td></tr><tr><td> 200毫升</td><td>火腿F-12 </td><td> N / A </td></tr></tbody></table></li><li>涡流轻轻ţO混合，分装10毫升，每管17×100无菌单元帽管。 </li><li>贮存于-80°C间</li><li>重要事项： <ul type="disc"><li>立即在使用前要解冻的NGF。 </li><li>前aliquoting NGF原液，预涂内墙组织培养塑料与血清蛋白吸管以减少NGF的坚持塑料。最简单的方法是要吸取的DMEM培养液的混合物，含有牛血清白蛋白的几次。 NGF的管冲洗几次与培养液混合物。 </li><li>新增的F-12最后，因为自由半胱氨酸可能会破坏胰岛素的二硫键。 </li><li>不冻结在体积大于10毫升，因为投诉警察课4晶体可能形成</li><li>不要冻结解冻C2的介质的2倍以上。 </li></ul></li></ol> 2。玻片的制备<ol><li>我们已经发现只精致的德国盖玻片工作在本协议一致（见表1推荐的源目录编号）。 </li><li>聚四氟乙烯（PTFE或聚四氟乙烯）盖衬垫（费舍尔目录＃09-911-765），加入75-100毫升70％硝酸至300毫升的玻璃标本瓶。 </li><li>戴手套，下降一成硝酸溶液盖玻片。补充足够的盖玻片上的罐子底部厚厚的一层不超过2-3盖玻片。 </li><li>轻轻摇晃容器，在室温轨道摇床集不超过185 RPM为6-8小时，盖玻片。 </li><li>在通风柜工作，精心调迁酸和处置根据当地的法律法规。超纯组织培养级水冲洗一次盖玻片，再加入75-100毫升水的容器。的盖玻片可能被漂洗多次，如果水变得浑浊。轻轻摇晃在室温下一套不超过185转的轨道摇床过夜。 </li><li>重复共3天以上的硝酸/组织培养级水每天序列。 </li><li>倒出决赛组织培养级水和补充足够的丙酮，几乎涵盖的盖玻片。 </li><li>倒出丙酮和妥善处理。 </li><li>盖玻片转移到150毫米的玻璃培养皿。补充足够的丙酮，几乎涵盖盖玻片。确保在单层盘的盖玻片。 </li><li>允许丙酮在通风柜一夜之间蒸发。 </li><li>在180°C时为1.5小时消毒烘干烤箱烘烤盖玻片。 </li></ol> 3。编制文化盖玻片<ol><li>解剖前的一天，加1盖玻片（如上所述准备）以及每24孔组织培养板使用无菌技术。每孔加入200μL无菌聚-D-赖氨酸溶液（100毫克/毫升在0.5 M Tris缓冲液，pH值8），并储存在4°C过夜板。 </li><li>一天的清扫，最好开始前的清扫，吸聚-D-赖氨酸的解决方案，并从每口井，每口井的5倍洗净用无菌组织培养级水。 </li><li> 200μL低血糖的DMEM培养液添加到每个板块和商店在37°C间孵化。约1小时，前电镀细胞，去除低血糖的DMEM培养液，每孔加250μLC2的介质。商店板在37℃下5％的CO 2，直到细胞准备是镀金。 </li></ol> 4。颈上神经节的解剖和隔离<ol><li>交感神经元的分离培养分离的颈上神经节从产前收获（上海建工）（胚胎19-21天）幼鼠通常准备，因为有较少的神经胶质细胞。然而，这个相同的协议，可以成功地用于产后早期（1〜3天）的幼鼠或围产期小鼠幼仔。 </li><li>安乐死通过CO 2吸入孕鼠。剃腹部皮肤用70％乙醇。在无菌条件下取出子宫角和子宫角放置在无菌的150毫米培养皿。避免剥离期间损坏的肠子或其他内脏器官大坝。 </li><li>培养皿放置到冰锅麻醉胚胎幼仔，幼仔。解剖胚胎的子宫角和羊膜。切断脊髓肩下沿中线小狗安乐死的小狗。这也切断腔静脉，从而降低了颈动脉出血在解剖的SCG。子宫角的一次解剖，在无菌莱博维茨的L-15培养基辅以青霉素和链霉素（或任何空气缓冲介质）的地方胚胎。 </li><li>与Sylgard半满150毫米培养皿的胚胎放在自己的背上。使用无菌20号针头，针的胸部和头部，轻轻hyperextending脖子上，这有利于解剖的SCG。 </li><li>使沿颈部正中切口在揭示颌下腺的锁骨水平。取下腺体使用罚款钳（ñO。 4。 5杜蒙）暴露肌肉层。 </li><li>使用罚款钳切断胸锁乳突肌锁骨附近。然后轻轻抬起和肩胛舌骨肌切开气管旁。此肌肉非常薄，所以要避免切割得太深，以避免底层的颈动脉撕裂和SCG。一旦删除这些肌肉层，颈动脉和迷走神经的应该是清晰可见。 </li><li>在SCG位于颈动脉分叉以下。使用罚款钳（第4号或5号），钝性解剖颈动脉双方组织了揭露节。用细镊子2，把握颈动脉的正上方，下方的SCG延髓和尾两端，分别删除的颈动脉，躺在以上SCG节以及在SCG本身。结状神经节，位于沿着这个发展阶段的上海建工，很可能也将在这一步中删除。结状神经节可以identifie其圆形的形状和大迷走神经从投影。这在于沿颈内动脉的分支之一。相比之下，上海建工杏仁状，在于直接下颈动脉分叉。放入无菌的35毫米文化菜含有L - 15培养基和抗生素的解剖组织。从所有幼仔进展到下一个步骤之前，上海建工。 </li><li>用细镊子，轻轻分开颈动脉和删除的任何其他组织在解剖的上海建工（尤其是迷走神经结状神经节）。用细镊子从节胶囊。这大大降低了文化中的非神经细胞的数目。神经节转移到另一个无菌的35毫米的菜，含L-15中。 </li><li>游离在SCG，删除的L-15中型和替换（终浓度为1毫克/毫升）2毫升胶原酶/ Dispase酶中的钙和镁汉克的平衡盐溶液（终浓度为5毫克/毫升）。 incubaTE 37°C时为40分钟。 </li><li>在SCG转移到无菌的15毫升锥形离心管，使量高达10毫升的L-15与BSA（终浓度为1-2毫克/毫升）的补充。在室温下离心5分钟，在200×Ğ。弃上清，再一次用L-15的补充与BSA。 </li><li>第二次洗涤后，删除所有的L-15和悬浮节〜2毫升的C2培养基。磨碎的细胞，用火抛光尖端弯曲吸管，确保，移液器涂BSA/L15前研磨，以最大限度地减少细胞粘到玻璃。磨碎轻轻的3-4倍。让大的团块，解决约1分钟，细胞悬液转移到15毫升锥形管。添加更多的C2媒体余下的上海建工和磨碎。让团块定居后，将细胞悬液从第二研磨后的第一次研磨收集的。重复此过程数次，直到该节主要是游离。 ðiscard第四研磨后的任何剩余的团块。 </li><li>从一个一窝幼鼠（一般为12-16幼仔）为细胞分离，使培养基中的细胞悬液总量8-10毫升加入额外C2的介质。用吸管轻轻悬浮细胞，取出一个小等分，以确定细胞密度。调整量达到2倍的文化所需的细胞密度，然后，一定要保持悬浮，分装250毫升细胞悬液，每孔细胞的细胞悬液。对于枝晶生长形态的研究，我们通常在低密度板细胞在24孔板（〜5×10 4细胞，每口井）。 </li><li>我们的实验室孵化SCG的文化在玻璃干燥器，而不是直接在二氧化碳培养箱。我们这样做是因为无血清培养基中生长的交感神经元略高CO 2浓度（约6.5％）执行。此外，我们不使用抗生素在OUR媒体似乎因为它抑制神经轴突生长和维持在干燥器的文化，如果它在发展文化的一个子集，以尽量减少污染扩散。使用这个系统，我们经常保持长达8个星期的上海建工文化。镀后，上海建工的文化放在瓷无菌水含在底部的玻璃干燥器干燥器板。约120毫升的CO 2注入干燥器前封它关闭，并放入一个孵化器集整个干燥器35.5°C。 </li></ol> 5。文化喂养和保养<ol><li>文化一般喂食，每周3次，电镀后24小时内发生的第一次媒体交流。使用无菌弯尖吸管，轻轻收回约一半，每口井的媒体。用干净的无菌弯尖吸管，更换新鲜的C2移除介质的体积。 </li><li>为了消除从文化的非神经细胞，抗有丝分裂剂细胞色素osine D-阿糖（Ara-c等，终浓度为1-2微米），通常被添加到在第一次媒体交流的培养基中，48小时（如，24小时后电镀）。这Ara-c等的浓度通常是足以消除所有非神经细胞。如果需要额外的ARA-C治疗，它的建议，这些被做了所有其他的喂养，以尽量减少对神经元的毒性作用。 </li></ol> 6。诱导与基底膜或者BMP-7的枝晶生长<ol><li>诱导树突状增长，基底膜或BMP-7添加到文化非神经细胞已被淘汰后，在文化的ARA-C水平显着降低。通常情况下，我们添加基底膜或BMP-7 在体外培养基上的第5天，但可以通过基底膜或在任何时间点在体外添加BMP-7诱导树突状增长。基底膜或BMP-7添加到C2的媒介和文化送入如上所述。 </li><li>基底膜和BMP诱导的树突状增长是时间和浓度依赖性。极大的影响，基底膜被添加到C2在终浓度为50-75微克/毫升; BMP-7在终浓度为50毫微克/毫升的C2培养基。基底膜浓度大于100微克/毫升或BMP-7浓度大于100 ng / ml时已经注意到造成神经毒性。树突状进程（形态学标准确定），成为明显的基底膜或BMP-7的最大有效浓度初步接触后约48小时。诱导树突生长健壮，文化喂养与BMP-7包含在每次喂养的介质。已取得相同的结果，使用20,21可比浓度骨形态发生蛋白2，4，5和6。 </li></ol> 7。 BMP-7-诱导的树突状生长的免疫组织化学分析<ol><li>当树突状树枝状的期望的程度已经达到了，文化是固定的，用4％多聚甲醛在0.1 M磷酸缓冲。最好的固定是chieved由4％多聚甲醛代替井一半的中等和重复上述步骤3-5倍，超过10-15分钟。 </li><li>漂洗文化取代一半的4％多聚甲醛磷酸盐缓冲液（PBS）和重复这一步骤三次超过10 - 15分钟。 </li><li>删除所有的PBS在室温下5分钟通透细胞添加200微升0.1％的Triton的PBS X-100的每口井。 </li><li>删除愿望TRITON解决方案，并添加200μL，每口井的阻断缓冲液（PBS 3-5％BSA和1％山羊血清补充）。孵育室温为20-30分钟。 </li><li>取出阻塞的缓冲区，并在封闭液中添加的主要抗体，微管相关蛋白2（MAP2）在1:2000稀释 - 1:5000。过夜室温或4°C孵育1小时的文化</li><li>删除主要的抗体溶液，洗超过15分钟的文化，在室温下用PBS三倍。 </li><li>在PBS稀释的第二抗体孵育培养补充室温在黑暗中，用1％的牛血清白蛋白在1-2小时。 </li><li>删除二级抗体溶液，并用PBS洗3次超过10-15分钟，在室温在黑暗中的文化。 </li><li>山玻璃盖玻片滑动细胞面朝下，在适当的pH值在水安装介质中二次抗体标记的荧光染料。在室温下保持的幻灯片，在一夜之间让安装介质干燥。商店幻灯片在4°C，直到成像。 </li><li>允许幻灯片前成像室温平衡。获得使用相衬显微镜萤光配备的荧光图像。通常情况下，整个一个单一的神经元的树突状乔木可以使用20X的目标和适当的过滤器捕获。 </li></ol> 8。代表结果脱离围产期RA上海建工交感神经元TS和生长在血清和神经节神经胶质细胞的情况下未能扩大MAP2的的阳性进程（图1），而是通常延长只有一个单一的轴突过程14,15。 BMP-7（图1）或基底膜暴露诱导形成的树突17,18形态学，生化和功能的标准，以满足众多的进程。基底膜或BMP-7枝促进活动是浓度和时间依赖性17,18,20。最大的枝晶生长的观察，使用浓度的基底膜之间的50和75微克/毫升或BMP-7 30至100毫微克/毫升，半最大效应通常是在BMP-7〜2毫微克/毫升的浓度观察。然而，在枝晶生长的显着变化，可以检测BMP-7低浓度高达300皮克/毫升。树突状基底膜或BMP-7的反应是相对缓慢，与&lt;50％的神经元，形成了暴露后24小时内第二个过程要么枝蔓-P<html> romoting代理。然而，最初的BMP-7曝光后3天之内，几乎所有的神经元的响应，以最大限度地有效基底膜或BMP-7浓度。每个神经元的树突的数量继续增加BMP-7的连续曝光，大多数发生变化，在第一个10天的治疗。 4周后，BMP-7治疗神经元通常有6-8主树突，表现出二级，三级和树突分支甚至四级17。树突状乔木的增加发生在轴突生长中的任何重大变化的情况下，可比树突状反应，可引起类似浓度骨形态发生蛋白2，4，5或6 20,21。树突生长也引起合作培养的交感神经元，与内源性神经节神经胶质细胞15,22;然而，树突的反应是在这些条件下慢得多。 JPG“/&gt; 图1。BMP-7促进培养的交感神经元的树突状增长 。非神经细胞的SCG的第2天48小时开始在体外的治疗与反分裂的文化被淘汰。 体外培养第5天开始，治疗要么控制介质（A）或补充BMP-7在50纳克/毫升（二）中等文化。在体外（骨形态发生蛋白治疗后5天）11天，文化与单克隆抗体免疫组化染色对微管相关蛋白，蛋白质主要存在于树突和神经元胞体。作为代表性的荧光显微照片显示，控制条件下生长的神经元缺乏树突MAP-2阳性树突过程（一）缺乏证明;相比之下，神经元暴露BMP-7（二）通常有几个锥形地图-2阳性树突。

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+autism.">The neurobiology of autism.</a> Brain Pathology (Zurich, Switzerland). 17, 434-447 (2007).</li><li>Zoghbi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurodevelopmental+disorders:+meeting+at+the+synapse.">Postnatal neurodevelopmental disorders: meeting at the synapse.</a> Science. 302, 826-830 (2003).</li><li>de Ruiter, J. P., Uylings, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+and+dendritic+analysis+of+fascia+dentata+granule+cells+in+human+aging+and+senile+dementia.">Morphometric and dendritic analysis of fascia dentata granule cells in human aging and senile dementia.</a> Brain Res. 402, 217-229 (1987).</li><li>Flood, D. G., Coleman, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+plasticity+in+normal+aging+and+decreased+plasticity+in+Alzheimer's+disease.">Hippocampal plasticity in normal aging and decreased plasticity in Alzheimer's disease.</a> Prog. Brain Res. 83, 435-443 (1990).</li><li>Jagadha, V., Becker, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+pathology:+an+overview+of+Golgi+studies+in+man.">Dendritic pathology: an overview of Golgi studies in man.</a> Can. J. Neurol. Sci. 16, 41-50 (1989).</li><li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+I.+Conditions+under+which+neurons+form+axons+but+not+dendrites.">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. I. Conditions under which neurons form axons but not dendrites.</a> Dev. Biol. 128, 324-336 (1988).</li><li>Tropea, M., Johnson, M. I., Higgins, 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+promote+dendritic+development+in+rat+sympathetic+neurons+in+vitro.">Glial cells promote dendritic development in rat sympathetic neurons in vitro.</a> Glia. 1, 380-392 (1988).</li><li>Lein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+extracellular+matrix+and+osteogenic+protein-1+on+the+morphological+differentiation+of+rat+sympathetic+neurons.">The effects of extracellular matrix and osteogenic protein-1 on the morphological differentiation of rat sympathetic neurons.</a> Int. J. Dev. Neurosci. 14, 203-215 (1996).</li><li>Lein, P., Johnson, M., Guo, X., Rueger, D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+protein-1+induces+dendritic+growth+in+rat+sympathetic+neurons.">Osteogenic protein-1 induces dendritic growth in rat sympathetic neurons.</a> Neuron. 15, 597-605 (1995).</li><li>Lein, P. J., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+and+a+basement+membrane+extract+have+different+effects+on+axonal+and+dendritic+outgrowth+from+embryonic+rat+sympathetic+neurons+in+vitro.">Laminin and a basement membrane extract have different effects on axonal and dendritic outgrowth from embryonic rat sympathetic neurons in vitro.</a> 136, 330-345 (1989).</li><li>Higgins, D., Lein, P. J., Osterhout, D. J., Johnson, M. I., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 177-295 (1991).</li><li>Beck, H. N., Drahushuk, K., Jacoby, D. B., Higgins, D., Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+protein-5+(BMP-5)+promotes+dendritic+growth+in+cultured+sympathetic+neurons.">Bone morphogenetic protein-5 (BMP-5) promotes dendritic growth in cultured sympathetic neurons.</a> BMC Neurosci. 2, 12-12 (2001).</li><li>Guo, X., Rueger, D., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+protein-1+and+related+bone+morphogenetic+proteins+regulate+dendritic+growth+and+the+expression+of+microtubule-associated+protein-2+in+rat+sympathetic+neurons.">Osteogenic protein-1 and related bone morphogenetic proteins regulate dendritic growth and the expression of microtubule-associated protein-2 in rat sympathetic neurons.</a> Neurosci. Lett. 245, 131-134 (1998).</li><li>Lein, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+induce+dendritic+growth+in+cultured+sympathetic+neurons+by+modulating+the+balance+between+bone+morphogenetic+proteins+(BMPs)+and+BMP+antagonists.">Glia induce dendritic growth in cultured sympathetic neurons by modulating the balance between bone morphogenetic proteins (BMPs) and BMP antagonists.</a> J. Neurosci. 22, 10377-10387 (2002).</li><li>Higgins, D., Lein, P., Osterhout, D., Johnson, M. I., Banker, G., Goslin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Culturing Nerve Cells. , 177-205 (1991).</li><li>Bruckenstein, D. A., Higgins, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+differentiation+of+embryonic+rat+sympathetic+neurons+in+tissue+culture.+II.+Serum+promotes+dendritic+growth.">Morphological differentiation of embryonic rat sympathetic neurons in tissue culture. II. Serum promotes dendritic growth.</a> Dev. Biol. 128, 337-348 (1988).</li></ol>

作者没有透露。

这种模式的优点包括：（一）文化是一个没有其他细胞类型17,23交感神经元的同质人口组成;（二）交感神经神经生长因子的要求建立的，它允许使用工作定义和界定媒体的各种媒介，与基板增长和维持交感神经元23（三）在这些文化中的神经元统一回应基底膜，BMP-7和民进党的其他骨形态发生蛋白的树突促进活动60A亚科17,18,20,21;及（d）基底膜或BMP诱导的树突形成细胞的存?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>盖玻片</td><td> bellco </td><td> 1943-10012 </td><td> 12毫米的圆形盖玻片＃1厚度，德国玻璃</td></tr><tr><td>弯曲的尖滴管</td><td> bellco </td><td> 1273-50003 </td><td></td></tr><tr><td>聚-D-赖氨酸</td><td>西格玛</td><td> P0899-100MG </td><td> 1毫克/毫升原液</td></tr><tr><td>蒸馏水</td><td> Gibco公司</td><td> 15230 </td><td></td></tr><tr><td>精细镊子杜蒙没有。 4和5 </td><td>的罚款科学工具（FST）的</td><td> 11252-30 11241-30 </td><td></td></tr><tr><td> 20表针</td><td>碧迪</td><td> 305176 </td><td></td></tr><tr><td>莱博维茨的L-15培养基</td><td> Invitrogen公司</tD&gt; <td> 11415 </td><td></td></tr><tr><td>胶原酶</td><td>沃辛顿</td><td> 4176 </td><td> 1毫克/毫升</td></tr><tr><td> Dispase酶</td><td>罗氏公司</td><td> 04942078001 </td><td> 5毫克/毫升</td></tr><tr><td>汉克的平衡盐溶液（钙，镁） </td><td> Invitrogen公司</td><td> 14185-052 </td><td></td></tr><tr><td> DMEM培养液低血糖</td><td> Invitrogen公司</td><td> 11885 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Invitrogen公司</td><td> 25030 </td><td> 20毫米</td></tr><tr><td>胰岛素/转/硒</td><td> Invitrogen公司</td><td> 51500-056 </td><td> 100X </td></tr><tr><td>脂肪酸的免费牛血清白蛋白</td><td> Calbiochem公司</td><td> 126609 </td><td></td></tr><tr><td>神经生长因子</td><td>哈伦生物制品</td><td> 5017 </td><td></td></tr><tr><td>火腿的F-12营养培养基（架F-12） </td><td> Invitrogen公司</td><td> 11765 </td><tD&gt; </td></tr><tr><td>阿糖胞苷</td><td>西格玛</td><td> C1768 </td><td></td></tr><tr><td>基底膜</td><td> BD公司</td><td> 356234 </td><td>避免反复冻融</td></tr><tr><td> BMP-7 </td><td> R＆D系统Curis公司</td><td> 345-BP BMP7的-07H </td><td>骨形态发生蛋白4，5和6的工作同样以及BMP-7，有时更容易获得商业</td></tr><tr><td>多聚甲醛</td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>的Triton X-100 </td><td>费舍尔</td><td> BP151-500 </td><td></td></tr><tr><td>小鼠抗大鼠MAP2的抗体</td><td>伯格单克隆抗体公司</td><td> SMI52 </td><td> 1/5000 </td></tr><tr><td>的Alexa Fluor 546山羊抗鼠</td><td> Invitrogen公司</td><td> A11003 </td><td> 1/10000 </td></tr><tr><td>安装媒体</td><td>延长抗淬灭分子探针套件</td> &lt;TD&gt; P7481 </td><td></td></tr></tbody></table>

inducing dendritic growth in cultured sympathetic neurons

树突状乔木的形状决定的总突触输入一个神经细胞可以接收1-3，并影响这些输入4-6的类型和分布。树突生长和可塑性的改变模式与7个实验模型中的神经行为功能受损，被认为有助于神经紊乱8-10和11-13的神经退行性疾病中观察到的临床症状。这种意见强调精确调节树突形态功能的重要性，并建议确定枝晶生长的机制，控制，不仅促进神经元的连接是如何在正常发展的规管的认识，但还可以提供各种神经系统疾病的新型治疗策略的洞察力。 枝晶生长的机理研究，将极大地促进了可用性Ø发模型系统，使神经元实验从一个国家，他们在不延长树突之一，在他们阐述树突状乔木与体内对口切换。从颈上神经节（SCG）围产期啮齿类动物中分离交感神经元的原代培养提供了这样一种模式。定义培养基中的血清和神经节神经胶质细胞的情况下培养时，交感神经元延长一个单一的过程，这是轴突，这个单极状态持续几周文化14,15个月。然而，无论是骨形态发生蛋白-7（BMP-7），16,17或18基底膜培养基除了引发这些神经元的延长，树突的形态，生化和功能的标准，满足多个进程。交感神经元脱离围产期啮齿动物的上海建工，并在特定条件下生长的同质人口的神经元19一致响应基底膜，BMP-7和其他骨形态发生蛋白decapentaplegic（民进党）和60A亚科17,18,20,21枝促进活动。重要的是，基底膜和BMP诱导的树突的形成发生在细胞的存活或轴突生长17,18变化的情况下。 在这里，我们描述了如何设置从上海建工围产期大鼠的交感神经元的分离培养，使他们顺应选择性枝促进基底膜或BMP的活动。

Watch this Scientific Journal Video about Inducing Dendritic Growth in Cultured Sympathetic Neurons at JoVE.com

Inducing Dendritic Growth in Cultured Sympathetic Neurons

我们描述了使用骨形态发生蛋白-7（BMP-7）或基底膜的协议，主要从围产期大鼠颈上神经节（SCG）分离交感神经元选择性诱导的树突状增长。

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

inducing-dendritic-growth-in-cultured-sympathetic-neurons

Research

JoVE Journal

Neuroscience

12.7K 次观看.  University of California, Davis. 我们描述了使用骨形态发生蛋白-7（BMP-7）或基底膜的协议，主要从围产期大鼠颈上神经节（SCG）分离交感神经元选择性诱导的树突状增长。

视频: Inducing Dendritic Growth in Cultured Sympathetic Neurons

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...