The authors thank researchers at the University of Iowa, Drs. Luis Tecedor, Ines Martins and Beverly Davidson for instructions and helpful comments regarding striatal cultures, and Drs. Kara Gordon, Nicole Bode and Pedro Gonzalez-Alegre for genotyping assistance and discussions. We also thank Dr. Eric Weyand (Animal Identification and Marking Systems) for helpful comments regarding tattooing, and Dr. Shutaro Katsurabayashi (Fukuoka University) for helpful comments regarding the mouse culture. This work was supported by grants from the American Heart Association, the Department of Defense (Peer Reviewed Medical Research Program award W81XWH-14-1-0301), the Dystonia Medical Research Foundation, the Edward Mallinckrodt, Jr. Foundation, the National Science Foundation, and the Whitehall Foundation (N.C.H.).

注：在本研究中进行的所有动物的程序批准了美国爱荷华大学的机构动物护理和使用委员会。 利用小鼠的纹身爪垫1.长期鉴定<ol><li>固定用爪子垫（足底面）一爪子对着实验者。持用拇指和食指的爪子。要小心，不要捏爪子。 注：稳定的固定化是很重要的，以确保该纹身颜料放入爪垫的真皮，并因此是永久性7。 </li><li>拭爪垫，用70％的乙醇在纱布海绵或拭子。 </li><li>涂抹护肤油以一个棉签，轻轻按压脚掌垫几次面的提示。只使用少量皮肤油;当存在时大量，它将阻止纹身颜料到达皮肤。 </li><li>浸纹身针的顶端到纹身油墨之前以纹身。使用清洁的，无菌和锋利纹身针，以减少疼痛和感染的可能性，并且还增加了纹身效率。 </li><li>而爪垫表面覆盖有皮肤油，垂直和轻轻按纹身针尖对在爪垫的中心的皮肤，和多次喷射墨。见表材料/设备关于电动纹身系统的信息。 </li><li>喷70％乙醇的纱布海绵，轻轻按压在足去除多余的纹身色素在皮肤表面，并检查纹身的质量。检查爪垫中间有一个黑暗，圆斑不同于正常皮肤色素沉着。如果纹身是没有黑暗，或方便查看足够大，重复之前的步骤纹身。 </li></ol> 2.使用快速PCR基因分型基因分型试剂盒新生小鼠<ol><li>消毒的小鼠尾巴用70％乙醇的远端，切割5mm或更小的尾部倾斜，并将其传输到用于PCR的类型的8条管的管。使用未使用刀片的每个小狗避免样本之间的交叉污染。或者，使用剪刀，但是在这种情况下，小心地和彻底地除去剩余的组织上用70％的乙醇中的刀片。检查出血。如果发生出血，施加压力的尾部用纱布海绵切割部分直到出血停止。 </li><li>添加200μl的DNA提取溶液到含有样品每个PCR管中。见表材料/设备及其有关的工具包组成和信息。 </li><li>将管条置于PCR热循环仪，并且使用下面的程序开始了DNA提取：1个循环的55℃10分钟，1个循环，在95℃进行10分钟，并保持在4℃。 注意：这是在稍后用于PCR相同的PCR热循环仪。 </li><li>经过DNA提取完成后，从热循环仪取出一管带次颠倒5次。 </li><li>转印各试样的4微升溶液（DNA提取物），以未使用的8管带的管，和与混合：10微升2X PCR预拌二，2微升混合正向＆反向引物（建议在0.5μM; 见表材料/设备的序列），和4微升不含核酸酶的H 2 O为每一个试样。使用台式离心机旋短暂（ 例如，3秒）。置于冰上在任何时候都管处理时除外。 </li><li>进行热循环。见 表材料/设备的热程序。 </li><li>检测扩增的DNA产物。装载从PCR（20微升）的总反应溶液直接进琼脂糖凝胶的孔中。加载分子量标记到一个单独的井。施加电场。 </li><li>取得频带的荧光图像在紫外光下。 </li></ol>鼠脑神经元3.原代培养S上胶质滋养层注意：程序脑解剖和细胞解离（3.1）是共同的所有后续步骤。对于小鼠胶质细胞培养的过程（3.2），大鼠神经胶质培养物（3.3），和小鼠的神经元培养物（3.4）分别描述之后。 <ol><li>脑解剖和细胞Dissocaiation <ol><li>牺牲一只小鼠或大鼠幼崽断头，迅速 ​​取出大脑，并放置到Hanks&#39;液（ 见表1对组合物）+ 20％在35毫米的培养皿的胎牛血清（FBS）（保持在冰上）。 </li><li>划破中线大脑分成两个半球。从大脑的每个半球除去感兴趣的区域（ 例如，大脑皮质，纹状体和海马）。从表面去除脑膜和主要血管。切开大脑区域到使用手术刀片4-10薄片。 </li><li>冲洗脑切片在一个15毫升的离心管中，一次机智ħHanks液+ 20％FBS中，然后3次，用Hanks液（ 即无血清）（所有4℃）。冲洗，加入5-10毫升溶液，让大脑切片沉降在管的底部，抽吸从顶部的溶液，并加入新鲜的溶液中。通过抽吸液完成清洗过程。 </li><li>滤波器2毫升含胰蛋白酶消化溶液（ 见表1对组合物）使用3毫升注射器和一个0.2微米的注射器过滤器，和直接添加滤液到含有脑片管。让胰蛋白酶消化进行在室温13分钟。 </li><li>通过吸大部分，然后加入7-10毫升Hanks液+ 20％FBS（4℃）的第一中和胰蛋白酶溶液。 </li><li>用Hanks冲洗脑切片，两次&#39;溶液+ 20％FBS中，然后三次用Hanks液（ 即无血清）（所有4℃）。通过吸取SOLUT完成漂洗程序离子。 </li><li>过滤2毫升解离溶液（ 见表1对组合物）使用3毫升注射器和一个0.2微米的注射器过滤器，和直接添加滤液到管与脑切片。 </li><li>机械轻轻研制过程10-20次，直到可见组织块消失分离的细胞。用棉花塞住，火抛光的巴斯德吸管，避免研磨过程中制作气泡。 </li><li>等待3分钟的小片安定下来。 </li><li>传送大部分的溶液（〜1.5毫升，留下一些溶液在底部）到包含3毫升Hanks液+ 20％FBS中的溶液（4℃）一个15毫升的离心管中，用棉花塞住，拒爆─抛光的巴斯德吸管。不转移的所有解决方案，因为包含任何沉积物的底部典型地导致在培养的劣化。 </li><li>离心13分钟到185克（〜1100转），在4℃。 </li><li>吸出上清液轻轻地，加入1毫升预热的电镀液（37℃），以沉淀，并轻轻吹打使用棉栓，火抛光的巴斯德吸管数次重悬它。 注：三种不同类型的电镀介质用于不同培养用途：镀介质-1小鼠的神经胶质细胞，镀介质-2小鼠的神经元，和电镀介质-3鼠神经元和神经胶质细胞（ 见表1的组合物） 。 </li><li>取出10微升该细胞悬浮液，用10微升的0.4％台盼蓝溶液混合，并测量活细胞密度，使用一个血球或自动细胞计数器。 </li></ol></li><li>鼠标胶质细胞培养<ol><li>洗盖玻片，以帮助建立小鼠细胞的健康文化。 <ol><li>沉浸盖玻片（圆形，直径12mm）在70％硝酸中的玻璃培养皿。使用铝箔避光，并将其放置在轨道摇床O / N。 </li><li>冲洗玻璃coversliPS在培养皿至少三次蒸馏水。沉浸其中在蒸馏水中。放在轨道摇床O / N的菜。 </li><li>干上了生物安全柜用Whatman150毫米滤纸盖玻片。 </li><li>高压灭菌盖玻片。 </li><li>放置盖玻片入24孔培养皿。 </li></ol></li><li>按照步骤1和2标签和基因型新生小鼠。 </li><li>获得来自小鼠幼仔的脑细胞，根据步骤3.1。 注：使用大脑皮层的是典型的准备鼠标胶质滋养层。然而，其他的大脑区域，例如海马和纹状体，将细胞密度的适当调整后的工作，由于不同的号码，并从不同的区域的细胞的产率。 </li><li>添加〜4毫升预热镀介质-1至最终细胞悬浮液（约1毫升）中。对于预热的培养基，放置在培养箱中的溶液（5％CO 2 -95％O 2，37℃）O / N，以允许的温度和pH值下保持稳定。 </li><li>细胞悬液转移到一个未涂覆的T25培养瓶中，用棉花塞住，火抛光的巴斯德吸移管。放置在所述培养箱中的烧瓶中。 </li><li>在第1天在体外 （DIV），冲洗培养的细胞在T25烧瓶中以电镀两次培养基-1（4℃）。将烧瓶放回培养箱。执行由抽吸烧瓶完全用巴氏吸管内侧的介质，加入约5毫升的新鲜培养基，并轻轻倾斜烧瓶几次以旋涡运动漂洗。 </li><li>在6-9 DIV，（ 即，一天胰蛋白酶消化前和电镀盖玻片上，在步骤3.2.8-3.2.13），放置100微升涂层材料与细胞外基质蛋白（见表材料/设备有关的评论涂层材料）上的盖玻片在培养皿，然后将培养皿在培养箱中。 </li><li>在7-10 DIV，当细胞是20-40％汇合（空间连续），trypsinize它们。 <ol><li>冲洗烧瓶一次，通过吸烧瓶内的所有的溶液并加入〜13毫升的Hanks&#39;液（4℃），轻轻倾斜烧瓶多次在一个旋涡运动，并完全吸出溶液。 </li><li>加入40微升DNA酶溶液（终浓度，750单位/ ml）到4ml胰蛋白酶-EDTA溶液，通过0.2μm的注射器过滤该溶液，并直接添加滤液至细胞在烧瓶中。 </li><li>让胰蛋白酶消化进行13分钟，在37℃的培养箱中培养。 </li><li>加入2毫升100％FBS（4℃）至烧瓶中和胰蛋白酶溶液。 </li><li>传送胰蛋白酶细胞用5至10毫升吸管一个15毫升的离心管中，加入Hank氏溶液+ 20％〜4毫升FBS的（4℃），离心〜185克和4℃下进行13分钟，并吸出上清液。 </li></ol></li><li>重悬沉淀在1ml预WÄRME的ð电镀介质-1。 </li><li>根据步骤3.1.13测量细胞的密度。 </li><li>吸出涂覆材料完全从玻璃盖玻片，并且板〜50微升的重新悬浮的神经胶质细胞的盖玻片上。 注：这些细胞将建立胶质滋养层。 </li><li>放置在培养皿中孵化盖玻片。 </li><li> 20-60分钟后，加入1 ml预热的镀介质-1向每个孔中，然后将培养皿放回培养箱中。注意：当镀介质被加入，这将是有帮助的，以使用另一个吸管压下盖玻片（在没有镀细胞）的外周，从而使盖玻片不会在培养基中漂浮。 </li><li>在1 DIV胶质饲养层（ 即，在玻璃盖玻片）的，用1ml更换介质预热镀介质-1，由抽吸所有的孔中的溶液中，然后用新鲜的培养基填充它。 </li><li>在2-3 DIV胶质饲养层的CEL时LS是80-100％汇合时，加有丝分裂抑制剂（10μl的5-氟-2&#39;-脱氧尿苷+尿苷的混合物;终浓度为81.2和204.8μM，分别地）到每个孔中，以抑制DNA复制，因此，以抑制神经胶质细胞的增殖。 </li><li>在7-9 DIV，当细胞是90-100％汇合（胶质饲养层上镀敷的神经元前1-2小时），以取代所述培养基预热镀介质-2（37℃）。 注：神经元将被镀在同一天，使用步骤3.4。 </li></ol></li><li>神经胶质文化<ol><li>涂覆T25培养瓶用相同的涂层材料。 注意：这是必要的，在步骤3.3.5以后除去非粘附细胞。 <ol><li>加入2.0毫升涂层材料至该烧瓶中，并将其放置在所述培养箱中的烧瓶中。 </li><li>后2-3小时，完全吸出涂​​覆材料，使得烧瓶中的地板变干燥。 </li></ol></li><li>获取细胞从大鼠幼仔，根据步骤3.1。 注：海马的CA3-CA1区用于制备大鼠胶质饲养层。然而，其他的大脑区域，例如大脑皮质和纹状体，将调整为在细胞密度由于不同数量和不同区域的细胞的产率的差异后工作。 </li><li>添加〜4毫升预热镀介质-3至最终细胞悬浮液（约1毫升）中。 </li><li>细胞悬液转移到涂覆的T25培养瓶中，用棉花塞住，火抛光的巴斯德吸移管。将烧瓶中的培养恒温箱（5％CO 2 -95％O 2，37℃）。 </li><li>在2-3 DIV，当细胞是20-40％汇合时，除去非粘附或弱粘附细胞，通过关闭盖子紧紧，摇动烧瓶大力〜10倍，抽吸该溶液，并加入4-5镀毫升介质-3（4℃）。重复此步骤一次。检查整个烧瓶地板的培养细胞（ 例如，用相差显微镜），以确认那些出现神经元（ 即那些的量，电池主体的外边缘出现相亮）被完全除去。根据需要重复该过程经常以除去这些细胞，然后将烧瓶返回到培养箱中。 注：实力和“奶昔”要求可能会有所不同个体之间的实验者的总数。重要的是不保持奶昔的总数的一组数字，但证实神经元样细胞被消除。 </li><li>在6-8 DIV，当细胞是90-100％汇合时，通道中的神经胶质细胞通过胰蛋白酶它们​​作为在步骤3.2.8。 </li><li>离心后，重悬沉淀在1ml电镀介质-3（4℃）中，加10镀介质-3（4℃）中的溶液中，胰蛋白酶消化细胞转移到未涂覆的T75烧瓶中，并将其培养在孵化器。 注：鼠的神经胶质细胞将传代两次;与此相反的小鼠的神经胶质细胞ARË代只有一次，因为他们成为多次传代后，不健康的。大鼠神经胶质细胞将传代以未涂覆的任何涂层材料T75烧瓶。该涂层允许大鼠神经胶质细胞生长太快而产生许多纤毛细胞（推定的室管膜细胞），这将在后面恶化的神经元的培养条件。 </li><li>在烧瓶中17-19 DIV胶质培养物（ 即，传代和1天胰蛋白酶消化前和通过步骤3.3.9-3.3.14镀到盖玻片后11天），将100微升的涂覆材料上未洗涤盖玻片（圆形，直径12mm）在培养皿中，然后将培养皿在培养箱中。 注：对于神经胶质文化的差异并没有注意到，在培养结果有或没有清洗的盖玻片。 </li><li>在烧瓶18-20 DIV胶质文化（ 即传代12天后），通过胰蛋白酶培养细胞准备胶质滋养层，根据STEP 3.2.8。 </li><li>在离心过程中，从玻璃盖玻片完全吸出涂​​料。 </li><li>离心后，重悬沉淀在1ml电镀介质-3（4℃）的，并测量细胞密度，根据步骤3.1.13。 </li><li>调整胶质密度至10 4活细胞/ ml，和板100微升胶质细胞悬浮在涂覆的玻璃盖玻片上。 注：这些细胞将建立胶质滋养层。 </li><li>放置培养皿用盖玻片放入培养箱为20-60分钟。 </li><li>加入1毫升镀介质-3（4℃）至各孔中，然后将培养皿放回培养箱中。 </li><li>在3-4 DIV胶质饲养层，当在盖玻片细胞是40-80％汇合时，加入1毫升预热的生长培养基（ 见表1对组合物），包含胞嘧啶β-D-阿拉伯呋喃糖苷（阿糖胞苷，4μM终浓度）来停止神经胶质细胞的增殖。板块神经s的7-9 DIV胶质饲养层时，细胞是60-80％汇合时，使用步骤3.4。 </li></ol></li><li>鼠标神经细胞<ol><li>按照步骤1和2标签和基因型新生小鼠。 </li><li>使用鼠标幼崽步3.1。 </li><li>调整的活细胞悬浮液的密度，以〜2.0×10 5细胞/ ml的用预温热镀介质-2-电镀对小鼠的神经胶质细胞，或使用电镀介质-3-电镀对大鼠神经胶质细胞。 </li><li>板中的细胞中的胶质饲养层上，通过轻轻地加入细胞悬浮液到每个培养基良好。注：细胞悬浮液中添加的量将通过细胞的目标数在培养井来确定。例如，板〜60微升细胞悬浮液以达到12000个细胞/孔。 </li><li>需要注意的是无解的变化是必要的神经元​​镀后。要小心，以避免从孔中的溶液蒸发超过文化的过程中，通过加湿里面Øf显示培养孵化器。 </li></ol></li></ol>

The author (Zhengmin Huang) is the president of EZ BioResearch LLC that produces reagents described in this article.

这里介绍的协议包括程序纹身标记/识别小鼠的基因分型，从尾部的提示小鼠，并为培养小鼠大脑的神经元密度低。在一个循环中使用6-8幼仔的实验中，这些过程通常需要约0.5小时，〜4小时和〜2小时，分别在共6-7小时的。这使得实际用于单个实验者完成所有从幼仔的出生到神经元培养物的电镀时所必需的程序 - 在少于单个工作日（除胶质饲养层之前制备的）。 纹身 </p...

遗传疾病的啮齿动物模型已被证明在建立正常蛋白质和核酸，以及在这些缺陷的病理生理后果的生理功能非常有用。举出缺陷型为参与关键的细胞功能的蛋白质的小鼠，以及病症如阿耳茨海默氏病的小鼠模型。然而，某些遗传操作可导致新生儿杀伤力不久或在出生后几天。在这些情况下，原代细胞培养物，因为活细胞可以从死亡前的胚胎或新生儿幼仔得到的重要工具，它们可以保持在至少几个星期的体外 ，并在这段时间内的早期神经元发育，可接着生化，功能和形态的实验。对于主要培养物，也可以是有益的直接制版的神经元密度低;这使得可以以可视化的个别胞体，树突，轴突轴和神经TERMINALS在高的空间分辨率。然而，神经元在低浓度的存活和分化通常要求它们被镀上一个胶质饲养层，共培养的神经胶质细胞在不存在与它们由胶质1空调物理接触，或在培养基中培养。 低密度的神经元培养物的对神经胶质饲养层的机构可以是依赖于快速和可靠的基因分型预先 - 在几个小时在对比几天。速度是特别重要的神经基因型需要被匹配到预先制备的胶质饲养层时。作为更实际的例子，它可能有必要决定哪些其中基因型的幼仔在产生培养物的使用，以优化实验的效率。 在这里，我们证明了工作的协议，已被用于快速，简单和可靠的小鼠基因分型在以前的出版物2-6。鼠标的尾巴，市售的试剂盒的使用。这个协议包括从组织单步提取核酸，并且既不需要一个核酸纯化步骤，也不使用终止缓冲液（“停止溶液”）。这种基因分型方法的可靠性是通过提出的一系列试验的结果时，差异被相对于该样品的起始量，动物的年龄和PCR扩增子的长度引入的说明。该套件提供了自动提取和可靠性的优势。 为求是全面，使用纹身长期鉴定基因型小鼠的也证实。纹身是通过将纹身油墨对皮肤的真皮（表皮下）实现7。的过程被描述为纹身新生儿或1日龄小鼠的爪垫，虽然纹身可以应用到身体的其他部位，如尾和脚趾，并以动画所有年龄段的ALS。另外，程序将被证明为在低密度电镀和培养小鼠的神经元，是根据不同类型的神经胶质饲养层2,8的优化制剂。 我们使用继承神经系统疾病DYT1肌张力障碍的遗传小鼠模型-造成的基因TOR1A突变为常染色体显性运动障碍（c.904_906delGAG / c.907_909delGAG; p.Glu302del / p.Glu303del）9。所编码的蛋白质，torsinA，属于（AAA +）蛋白质家族，其成员通常执行伴侣样的功能，帮助在“与不同的细胞活性相关的ATP酶”：蛋白质折叠，蛋白质复合物的拆卸，膜运输，和囊泡融合10-13。该突变导致一个在框内缺失谷氨酸的密码子的，并可能导致“早发性全身性肌张力障碍分离的”14,15的表现。但是，路径负责这种疾病ophysiological机制仍然知之甚少。在一个敲入小鼠模型中，该突变体等位基因是TOR1A tm2Wtd，以下简称TOR1AΔE提及。杂ΔE-torsinA基因敲除小鼠是可行和基因模仿人类患者的肌张力障碍DYT1，而纯合子基因敲除小鼠出生后16,17死，与延迟到出生后死亡受遗传背景的18。纯合敲除小鼠的早期死亡的必要条件是动物两者的基因分型，并建立的神经元培养物的迅速完成。作为分型的另一个例子，Tfap2a（转录因子AP-2α，活化增强子结合蛋白2α）将被使用。由该基因编码的蛋白质是在调节多种细胞过程，诸如增殖，分化，存活和凋亡19重要。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>REAGENTS - tattooing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Machine Cleanser</td>
			<td>Animal Identification and Marking Systems, Inc.</td>
			<td>NMCR3</td>
			<td>This is used to clean the needles and the holder after tattooing.</td>
		</tr>
		<tr>
			<td>Machine Drying Agent</td>
			<td>Animal Identification and Marking Systems, Inc.</td>
			<td>NDAR4</td>
			<td>This is used to dry the needles and holder after cleaning.</td>
		</tr>
		<tr>
			<td>Neonate Tattoo Black Pigment</td>
			<td>Animal Identification and Marking Systems, Inc.</td>
			<td>NBP01</td>
			<td></td>
		</tr>
		<tr>
			<td>Skin Prep Applicator</td>
			<td>Animal Identification and Marking Systems, Inc.</td>
			<td>NSPA1</td>
			<td>Q-tip.</td>
		</tr>
		<tr>
			<td>Skin Prep solution</td>
			<td>Animal Identification and Marking Systems, Inc.</td>
			<td>NSP01</td>
			<td>This reagent delivers a thin layer of oil that enhances the efficiency of tattooing and prevents tattoo fading, by (information from vendor): 1) preventing non-tattooed skin from being stained temporarily, thereby allowing the quality of a paw pad tattoo to be easily evaluated before the pup is returned to its home cage &#8211; the stained skin surface can be confused with the tattooed skin, 2) reducing skin damage during tattooing &#8211; softening the skin and lubricating the needle will help the needle penetrate the skin without causing skin damage, and 3) preventing molecular oxygen from entering the skin, thereby reducing inflammatory responses to reactive oxygen species that can be generated.</td>
		</tr>
		<tr>
			<td>REAGENTS - genotyping</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EZ Fast Tissue/Tail PCR Genotyping Kit (Strip Tube Format)</td>
			<td>EZ BioResearch LLC</td>
			<td>G2001-100</td>
			<td></td>
		</tr>
		<tr>
			<td>2X PCR Ready Mix II</td>
			<td>EZ BioResearch LLC</td>
			<td>G2001-100</td>
			<td>A red, loading dye for electrophoresis is included in the 2X PCR Ready Mix solution.</td>
		</tr>
		<tr>
			<td>Tissue Lysis Solution A</td>
			<td>EZ BioResearch LLC</td>
			<td>G2001-100</td>
			<td>Prepare DNA Extraction Solution by mixing 20 &#181;l of Tissue Lysis Solution A and 180 &#181;l of Tissue Lysis Solution B per specimen.</td>
		</tr>
		<tr>
			<td>Tissue Lysis Solution B</td>
			<td>EZ BioResearch LLC</td>
			<td>G2001-100</td>
			<td>Prepare DNA Extraction Solution by mixing 20 &#181;l of Tissue Lysis Solution A and 180 &#181;l of Tissue Lysis Solution B per specimen.</td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>VWR</td>
			<td>BDH 3092</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose optimized grade, molecular biology grade</td>
			<td>rpi</td>
			<td>A20090-500&#160;</td>
			<td>We use 2% agarose gels in TAE buffer containing the SYBR Safe DNA gel stain (diluted 10,000-fold) or ethidium bromide (0.5 &#181;g/ml gel volume).</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E7637-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid, disodium salt dihydrate (EDTA)</td>
			<td>Fisher</td>
			<td>BP120-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtered Pipet Tips, Aerosol-Free, 0.1-10 &#181;l</td>
			<td>Dot Scientific Inc</td>
			<td>UG104-96RS&#160;</td>
			<td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td>
		</tr>
		<tr>
			<td>Filtered Pipet Tips, Premium Fit Filter Tips, 0.5-20 &#181;l</td>
			<td>Dot Scientific Inc</td>
			<td>UG2020-RS</td>
			<td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td>
		</tr>
		<tr>
			<td>Filtered Pipet Tips, Premium Fit Filter Tips, 1-200 &#181;l</td>
			<td>Dot Scientific Inc</td>
			<td>UG2812-RS</td>
			<td>Use pipette tips that are sterile and free of DNA, RNase and DNase. For all steps involving DNA, use filtered pipette tips to avoid cross-contamination.</td>
		</tr>
		<tr>
			<td>Molecular weight marker, EZ DNA Even Ladders 100 bp</td>
			<td>EZ BioResearch LLC</td>
			<td>L1001</td>
			<td>We use either of these three molecular weight markers.</td>
		</tr>
		<tr>
			<td>Molecular weight marker, EZ DNA Even Ladders 1000 bp</td>
			<td>EZ BioResearch LLC</td>
			<td>L1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular weight marker, TrackIt, 100 bp DNA Ladder</td>
			<td>GIBCO-Invitrogen</td>
			<td>10488-058</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes, 8-tube strips with individually attached dome top caps, natural, 0.2 ml&#160;</td>
			<td>USA Scientific</td>
			<td>1402-2900</td>
			<td>Use tubes that are sterile and free of DNA, RNase and DNase. An 8-tube strip is easy to handle and to group the specimens than individual tubes.</td>
		</tr>
		<tr>
			<td>PCR tubes, Ultraflux Individual&#160;</td>
			<td>rpi</td>
			<td>145660</td>
			<td>Use tubes that are sterile and free of DNA, RNase and DNase.</td>
		</tr>
		<tr>
			<td>Seal-Rite 0.5 ml microcentrifuge tube, natural</td>
			<td>USA Scientific</td>
			<td>1605-0000</td>
			<td>Use tubes that are sterile and free of DNA, RNase and DNase.</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain * 10,000x concentration in DMSO</td>
			<td>GIBCO-Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>rpi</td>
			<td>T60040-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers for amplifying Tor1a gene in &#916;E-torsinA knock-in mice</td>
			<td></td>
			<td></td>
			<td>5&#39;-AGT CTG TGG CTG GCT CTC CC-3&#39; (forward) and 5&#39;-CCT CAG GCT GCT CAC AAC CAC-3&#39; (reverse) (reference 18). These primers were used at a final concentration of 1.0 ng/&#181;l (~0.16 &#181;M) (reference 2).</td>
		</tr>
		<tr>
			<td>Primers for amplifying Tfap2a gene in wild-type mice</td>
			<td></td>
			<td></td>
			<td>5&#39;-GAA AGG TGT AGG CAG AAG TTT GTC AGG GC-3&#39; (forward), 5&#39;-CGT GTG GCT GTT GGG GTT GTT GCT GAG GTA-3&#39; (reverse) for the 498-bp amplicon, 5&#39;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#39; (forward), 5&#39;-AGA CAC TCG GGC TTT GGA GAT CAT TC-3&#39; (reverse) for the 983-bp amplicon, and 5&#39;-CAC CCT ATC AGG GGA GGA CAA CTT TCG-3&#39; (forward), 5&#39;-ACA GTG TAG TAA GGC AAA GCA AGG AG-3&#39; (reverse) for the 1990-bp amplicon. These primers are used at 0.5 &#181;M.</td>
		</tr>
		<tr>
			<td>REAGENTS - cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100MG</td>
			<td>See comments section of uridine for more information.</td>
		</tr>
		<tr>
			<td>B-27 supplement</td>
			<td>GIBCO-Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dishes 35 x 10 mm Dishes, Tissue Culture-treated</td>
			<td>BD falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flasks, T25, Tissue Culture-treated, Canted-neck, plug-seal cap, 25 cm2 Growth Area, 70 ml</td>
			<td>BD falcon</td>
			<td>353082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Flasks, T75, Tissue Culture-treated, Canted-neck, vented cap, 75 cm2 Growth Area, 250 ml</td>
			<td>BD falcon</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tube, polypropylene, 15 ml</td>
			<td>BD falcon</td>
			<td>352095</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess (cell number counter) chamber slides</td>
			<td>GIBCO-Invitrogen</td>
			<td>C10312</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-Arabinofuranoside hydrochloride (Ara-C hydrochloride)</td>
			<td>Sigma-Aldrich</td>
			<td>C6645-100mg</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose (Dextrose) anhydrous, SigmaUltra, 99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>G7528-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dish, Petri glass 100 x 15 mm</td>
			<td>Pyrex</td>
			<td>3160-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>GIBCO-Invitrogen</td>
			<td>15230-147</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase Type II</td>
			<td>Sigma-Aldrich</td>
			<td>D4527-200KU</td>
			<td>Stock solution is prepared at 1500 units/20 &#956;l = 75000 units/ml in distilled water.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX, pyruvate</td>
			<td>GIBCO-Invitrogen</td>
			<td>10569-010, 500 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast PES Filter Unit, 250 ml, 50 mm diameter membrane, 0.2 &#181;m Pore Size</td>
			<td>Nalgene</td>
			<td>568-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast PES Filter Unit, 500 ml, 90 mm diameter membrane, 0.2 &#181;m Pore Size</td>
			<td>Nalgene</td>
			<td>569-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBCO-Invitrogen</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip, 12 mm Round, thickness 0.09&#8211;0.12 mm, No. 0</td>
			<td>Carolina</td>
			<td>633017</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>GIBCO-Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salts</td>
			<td>Sigma-Aldrich</td>
			<td>H2387-10X</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES, &#8805;99.5% (titration)</td>
			<td>Sigma-Aldrich</td>
			<td>H3375-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 37%, A.C.S reagent</td>
			<td>Sigma-Aldrich</td>
			<td>258148-100 ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I5500-250 mg</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate, MgSO4&#8226;(7H2O), BioUltra, &#8805;99.5% (Fluka)</td>
			<td>Sigma-Aldrich</td>
			<td>63138-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix solution, Phenol Red-Free</td>
			<td>BD Biosciences</td>
			<td>356237</td>
			<td>This is the coating material for coverslips and flasks. 1) To prepare it, thaw the Matrigel Basement Membrane Matrix solution on ice, which usually takes ~1 day. Using a pre-cooled pipette, aliquot the thawed solution into pre-cooled T25 flasks on ice, and store the flasks at -20&#176;C. To prepare the working Matrigel solution, thaw the aliquotted Matrigel in a flask on ice, dilute 50-fold by adding pre-cooled MEM solution and keep the diluted solution at 4&#176;C. It is important to pre-cool all cultureware and media that come into contact with Matrigel, except during and after the coating of coverslips, to prevent it from prematurely forming a gel. 2) To coat the glass coverslips or culture flasks with Matrigel, apply the Matrigel solution to the surface. Before plating cells, it is important to completely dry up the surface. For this purpose, it might be helpful to aspirate Matrigel during the cellular centrifugation immediately before plating the cells and to allow enough time for drying.</td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>GIBCO-Invitrogen</td>
			<td>51200-038</td>
			<td></td>
		</tr>
		<tr>
			<td>MITO+ Serum Extender, 5 ml</td>
			<td>BD Biosciences</td>
			<td>355006</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Plates, Tissue Culture-treated 24-well plate</td>
			<td>BD falcon</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell Plates, Tissue Culture-treated 6-well plate</td>
			<td>BD falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium (1X), liquid</td>
			<td>GIBCO-Invitrogen</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric Acid</td>
			<td>VWR</td>
			<td>bdh 3044</td>
			<td></td>
		</tr>
		<tr>
			<td>NS (Neuronal Supplement) 21&#160;</td>
			<td>prepared in the lab</td>
			<td></td>
			<td>Source: reference 69</td>
		</tr>
		<tr>
			<td>Pasteur pipets, 5 &#190;&#8221;&#160;</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>Use this cotton-plugged 5 &#190;&#8221; Pasteur pipette for cellular trituration. Fire-polish the tip beforehand to smooth the cut surface and to reduce the internal diameter to 50-80% of the original. Too small a tip will disrupt the cells and reduce cell viability, but too large a tip will decrease the efficiency of trituration.</td>
		</tr>
		<tr>
			<td>Pasteur pipets, 9&#8221;&#160;</td>
			<td>Fisher</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl), SigmaUltra, &#8805;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>P9333-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 2 ml</td>
			<td>BD falcon</td>
			<td>357507</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 5 ml&#160;</td>
			<td>BD falcon</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 10 ml</td>
			<td>BD falcon</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 25 ml</td>
			<td>BD falcon</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 50 ml&#160;</td>
			<td>BD falcon</td>
			<td>357550</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3, Sodium hydrogen carbonate), SigmaUltra, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>S6297-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl), SigmaUltra, &#8805;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>S7653-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH), pellets, 99.998% trace metals basis</td>
			<td>Sigma-Aldrich</td>
			<td>480878-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic heptahydrate (Na2HPO4&#8226;(7H2O)), &#8805;99.99%, Aldrich</td>
			<td>Sigma-Aldrich</td>
			<td>431478-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose, SigmaUltra, &#8805;99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>S7903-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, sterile, 0.2 &#181;m</td>
			<td>Corning</td>
			<td>431219</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 3 ml</td>
			<td>BD falcon</td>
			<td>309585</td>
			<td></td>
		</tr>
		<tr>
			<td>Transferrin, Holo, bovine plasma</td>
			<td>Calbiochem</td>
			<td>616420</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue stain, 0.4%</td>
			<td>GIBCO-Invitrogen</td>
			<td>T10282</td>
			<td>This is used for counting live/dead cells. Renew an old trypan blue solution if it is re-used many times (e.g. several times a week for several weeks), because it will form precipitates and result in erroneous readouts of cellular density.</td>
		</tr>
		<tr>
			<td>Trypsin, type XI</td>
			<td>Sigma-Aldrich</td>
			<td>T1005-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution, 0.25%&#160;</td>
			<td>GIBCO-Invitrogen</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3003-5G</td>
			<td>Stock solution is prepared at 50-mg 5-fluoro-2&#39;-deoxyuridine and 125-mg uridine in 25 ml DMEM (8.12 and 20.48 mM, respectively).</td>
		</tr>
		<tr>
			<td>REAGENTS - immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody, rabbit polyclonal anti-MAP2</td>
			<td>Merck Millipore</td>
			<td>AB5622</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody, mouse monoclonal anti-GFAP cocktail</td>
			<td>Merck Millipore</td>
			<td>NE1015</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid genotyping of animals followed by establishing primary cultures of brain neurons

哺乳动物神经元的形态和功能的高分辨率分析往往需要个体动物随后的神经元的原代培养的分析的基因分型。我们描述了一套程序：标记新生小鼠进行基因分型，快速基因分型，并建立从这些小鼠脑的神经元的低密度培养物。个别小鼠通过纹身，其允许长期持久的识别到成年标记。基因分型通过所述协议是快速和有效的，并且允许进行核酸具有良好的可靠性的自动提取。这是根据情况下有足够的时间对常规的基因分型是不可用， 例如 ，是有用的，在于从新生致死遭受小鼠。在低浓度，这使得成像实验以高的空间分辨率产生原代神经元培养物。这种培养方法需要胶质滋养层的准备之前，神经元电镀。在protocol是其全部施加到运动障碍DYT1肌张力障碍（ΔE-torsinA敲除小鼠）的小鼠模型中，与神经元培养物从这些小鼠的海马，大脑皮质和纹状体制备。该协议可以被应用到小鼠与其他的基因突变，以及对其他物种的动物。此外，可用于分离子项目的协议的各个组件。因此，该协议将有广泛的应用，不仅在神经科学，而且在生物科学和医学等领域。

作为该协议的应用的一个例子，有代表性的结果示于通过纹身，可靠的基因分型的各种实验条件下标记的小鼠，并且对神经胶质饲养层建立初级神经元培养物。 纹身 新生幼仔被标记上（ 图1中“新生儿”），使用纹身系统的爪垫。标签保持3周清晰可见（&#39;3周龄&#39;）和32周龄（&#39;32周龄&#39;）。个别小鼠可通过在四只爪子（编...

Watch this Scientific Journal Video about Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons at JoVE.com

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

我们描述了标签和基因分型新生小鼠，并从中产生的主要神经文化的过程。基因分型是快速，有效和可靠的，并且允许进行自动核酸提取。这是为neonatally致死小鼠和其文化，需要事先完成基因分型的特别有用的。

动物的基因分型快速其次是建立大脑神经元原代培养

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

rapid-genotyping-animals-followed-establishing-primary-cultures-brain

Research

JoVE Journal

Neuroscience

16.2K 次观看.  University of Iowa Carver College of Medicine. 我们描述了标签和基因分型新生小鼠，并从中产生的主要神经文化的过程。基因分型是快速，有效和可靠的，并且允许进行自动核酸提取。这是为neonatally致死小鼠和其文化，需要事先完成基因分型的特别有用的。 

视频: Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

检测蛋白质棕榈酰化培养海马神经元通过免疫沉淀和酰基-生物素交易所（ABE）

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

原代海马神经元的磷酸钙转染

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Rapid and Specific Immunomagnetic Isolation of Mouse Primary Oligodendrocytes

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of oligodendroglia as well as the biology of demyelinating diseases such as multiple sclerosis and Pelizaeus-Merzbacher-like disease (PMLD). Here, we present a simple and efficient selection method for the immunomagnetic isolation of stage three O4+ preoligodendrocytes cells from neonatal mice pups. Since immature OL constitute more than 80% of the rodent-brain white matter at postnatal day 7 (P7) this isolation method not only ensures high cellular yield, but also the specific isolation of OLs already committed to the oligodendroglial lineage, decreasing the possibility of isolating contaminating cells such as astrocytes and other cells from the mouse brain. This method is a modification of the techniques reported previously, and provides oligodendrocyte preparation purity above 80% in about 4 h.

We describe the immunomagnetic isolation of primary mouse oligodendrocytes, which allows the rapid and specific isolation of the cells for in vitro culture.

小鼠初级少突胶质的快速特异磁分离

The efficient and robust isolation and culture of primary oligodendrocytes (OLs) is a valuable tool for the in vitro study of the development of ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...