We thank Michael Candlish for critical comments on the manuscript. This project was supported by the Deutsche Forschungsgemeinschaft grants BO1743/6 and SFB/TRR 152 P11 and Z02 to Ulrich Boehm.

注:伦理学声明:涉及动物对象的程序批准了汉堡萨尔州的大学和大学的动物福利委员会。 1.制备和胚胎组织的固定<ol><li>安排所有解剖出胚胎和牺牲动物前准备组织随后固定解决方案所需的设备。 注:始终制备新鲜的4％多聚甲醛（PFA）溶液（4％PFA的0.1M磷酸盐缓冲盐水（PBS），调节pH至7.4）。 </li><li>安乐死定时怀孕的雌性小鼠与道德批准的程序。 </li><li>转移到小鼠的平台。湿用70％乙醇腹部的腹侧和使用锋利的剪刀，以暴露腹腔的垂直切口。 </li><li>确定胚胎链拉出，用钝钳，将其放入冰冷的PBS。 </li><li>请从单个胚胎胚胎链用细剪刀和镊子。除去多余的组织周围的胚胎和在冰冷的PBS冲洗。 </li><li>取一小尾活检（转移胚胎到PFA之前）和孵化它的性别鉴定和示踪等位基因的基因分型PCR尾巴裂解液。 </li><li>从胚胎开始在最外侧面，并分别传送每个胚胎成含有冰冷的4％PFA在冰上在振荡器上的管。 </li><li>浸泡胚胎E13.5岁或以下1.5小时冰上持续振荡。浸泡胚胎年龄E14.5 E15.5和在冰上与持续振荡2.5小时。 </li><li>对于胚胎年龄E16.5以上，斩首和浸泡头PFA解决方案之前，除去外皮。 E16.5胚胎的头可以浸泡在冰与持续振荡4.5小时。 </li><li>经过适当的固定，用冰冷的PBS洗胚胎三次。直到它们下沉胚胎转移到30％蔗糖的PBS溶液在4℃下底部。 </li></ol> 2.冷冻<ol><li>安排所有在开始冻结过程之前所需的材料:低温模，低温手套，轻量级昆虫学镊子，记号笔和最佳切削温度化合物（OCT） </li><li>标签的低温模具具有永久耐酒精标记（提组织，日期和基因型的方向）。准备粉碎的干冰和100％乙醇的溶雪在冰桶。放置内含有异戊烷的玻璃烧杯中。等待10分钟为异戊烷冷却下来。 </li><li>同时除去来自蔗糖溶液的组织，使用实验室抹布擦去组织周围多余的蔗糖。转移组织成预标记的冷冻模具具有足够的OCT以覆盖组织。避免任何气泡在OCT;特别是在接近组织。 </li><li>使用轻量级昆虫学镊子，避免组织损伤定位在OCT组织。首先转移低温冷冻摩尔D装入预先冷却的异戊烷浴。不要溅进异戊烷华侨城，因为它不会冻结正确。包裹在铝箔和储存的样品在-80℃下。 </li></ol> 3. Cryosectioning <ol><li>切用低温恒温器在五大系列"14微米薄连续切片和收集SuperFrost®加玻片进行免疫荧光（IF）的分析。存储的幻灯片在-80℃直到使用。 </li></ol> 4.示踪可视化使用高通量筛选（TSA）协议<ol><li>备所需的染色缓冲液和试剂。总是新鲜准备的Tris-氯化钠吐温（TNT）缓冲区上使用的当天。 <ol><li>准备使用1M的Tris-HCl为100毫升pH为7.5，将30ml的5M NaCl和DDH 2 O的高达1 L的终体积加入500微升吐温20的使用1毫升吸管的TNT缓冲液中。添加吐温20缓慢并冲洗移液器上下几次，以确保所有的吐温20处于解决方案。 </li><li>制备的Tris-NaCl的阻断用100毫升的1摩尔的Tris-HCl的pH为7.5，将30ml的5M NaCl和DDH 2 O的（TNB）缓冲液达1 L的终体积加入5克封闭试剂（设置有盒）慢慢以小增量的缓冲区，同时搅拌。逐渐加热该溶液至55℃，并连续搅拌，完全溶解封闭试剂（这不应超过30-60分钟需要更长的时间）。以实现均匀加热用水浴在55℃。该解决方案将出现乳白色。在使用前使溶液至室温。等分试样并储存在-20℃下长期贮存。 </li><li>重组生物素酪酰胺试剂（由试剂盒提供）在分子生物学/ HPLC级的DMSO。取决于哪个试剂盒用于DMSO中的适当量可以变化。储存原液于4℃。 可用于电路分析部分从动物杂合的各自的Cre和R 26等位基因:注。采用Sections从同窝承载1 的Cre或1 R 26等位基因，分别作为阴性对照。治疗阴性对照幻灯片酷似双重杂合子动物幻灯片。此外，包括双重杂合子的动物一个控制幻灯片，但离开了TSA试剂（非放大控制）。 </li></ol></li><li>使用锋利的手术刀清新去除多余华侨城周围的组织切片，并标记周围的部分边界用PAP笔。 </li><li>干燥载玻片在室温下2-3分钟（RT）。洗幻灯片在RT 3倍与TNT，每次5分钟。在0.3％的孵育载玻片在室温进行30分钟，在冰冷的甲醇中的 H 2 O 2溶液以猝灭内源性过氧化物酶的活性。 </li><li>洗涤3次，用PBS进行5分钟，每在RT。孵育在0.5％的Triton-X 100 10分钟的PBS透化组织。洗幻灯片在RT 3倍与TNT，每次5分钟。 </li><li>用TNB块（封闭溶液，每滑动200-300微升）30分在RT在加湿室中。确保所有的部分都完全覆盖TNB。不要让幻灯片干的步骤之间。 </li><li>流掉过量封闭缓冲液，并应用主抗血清识别的示踪剂（1:1000山羊抗WGA对BL，1:15000的兔抗GFP为GTT）在RT或过夜，在4℃稀释在TNB 2小时。一定要使用足够的量来完全覆盖组织切片（每张幻灯片200-300微升）。 注:此处推荐的主要抗血清稀释可能要进行调整。 </li><li>洗幻灯片在RT 3倍与TNT每个搅拌5分钟。 </li><li>孵育生物素化的第二抗血清的部分识别第一抗血清的主机（1:500的马抗山羊IgG抗WGA，1:5000山羊抗兔IgG抗GFP在TNB 1小时在RT）。 </li><li>洗3倍与TNT在室温每搅拌5分钟。孵育在1:100链霉亲（SA）标记的辣根过氧化物酶（SA-HRP，提供WiTH试剂盒）在TNB在室温30分钟，在湿润的腔室中。洗幻灯片在RT 3倍与TNT，每次5分钟。同时解冻TSA。 </li><li> TSA稀释1:50 TSA放大稀释剂（试剂盒提供;使用TSA套件BL的可视化和TSA +套件GTT）。孵育组织切片在稀TSA用于精确10分钟，在室温。 TSA稀释使用前，在以往的洗涤步骤理想情况下，不使用任何剩余的稀释TSA试剂未来的实验;始终准备好新鲜。 </li><li>洗幻灯片在RT 3倍与TNT每个搅拌5分钟。孵育，用1:500的AlexaFluor®546链霉抗偶联物在TNB 30分钟，在室温。洗幻灯片在RT 3倍与TNT每个搅拌5分钟。 </li><li>孵育在PBS中5％的双苯甲亚胺溶液在RT核染色10分钟。洗幻灯片在RT 3倍与TNT每个搅拌5分钟。安装与Fluromount G和进行表面荧光或共聚焦显微镜。 <em&gt;故障排除:示踪剂转移功效将取决于ROSA26基因座中产生细胞（由所使用的酶Cre驱动线确定的）的表达水平与神经电路上进行分析（神经元产生的示踪物，收敛等的例如号码）。因此这里推荐初级抗血清稀释度可能需要进行调整，以防止低或过量的信号。背景应该总是使用适当的控制幻灯片（见上文）进行分析。 </li></ol> 5.τlacZ染色的胚胎切片<ol><li>备所需的染色缓冲液和试剂。使用之前，请务必刚准备LacZ的缓冲液C。 <ol><li>通过加入40克X-gal的1毫升70％二甲基甲酰胺（DMF）中制备的5-溴-4-氯-3-吲哚 - β-D-吡喃半乳糖苷（X-gal的）储备溶液（40毫克/毫升）。用铝箔覆盖，并储存在-20℃下长期贮存。 </li><li>准备硝基四氮唑蓝（NBT）ST玉珠溶液（50毫克/毫升）加入50克的NBT到1ml 70％的DMF。用铝箔覆盖，并储存在-20℃下长期贮存。 </li><li>制备的LacZ定影液用80微升25％的戊二醛，5毫升4％PFA中，20微升的1M MgCl 2的，加入100μl的500mM EGTA，加入1ml的1M磷酸盐缓冲液pH 7.4和DDH 2 O的最多至最终体积为10ml。 </li><li>制备的LacZ缓冲液A用1ml的1M的MgCl 2，5毫升的500mM EGTA 50毫升双蒸的1M磷酸盐缓冲液pH 7.4和2 O最多为500毫升的最终体积。 </li><li>制备用1ml的1M的的MgCl 2，5毫升的500mM EGTA，毫升1％的5脱氧胆酸钠，加入100ml的10％的NP-40，将50ml的1M磷酸盐缓冲液pH 7.4和DDH 2 O的 LacZ的缓冲液B最多为500毫升的最终体积。 </li><li>制备的LacZ缓冲液C中加入10微升的NBT原液和12.5微升X-gal的原液到1ml的LacZ缓冲液B的制作新鲜临使用前和用铝箔覆盖。 </li></OL&gt; </li><li>干的幻灯片在室温至少10-15分钟（同时准备用PAP笔幻灯片）。 </li><li>固定部与LacZ的固定剂2分钟，在室温下，在化学通风橱内的潮湿室中。用PBS洗3次，补充了2mM的MgCl 2。 </li><li>冲洗用的LacZ缓冲A.幻灯片与洗净的LacZ幻灯片缓冲A 3次，在室温每次10分钟。每次5分钟，洗2次，用LacZ的缓冲液B。孵育在LacZ的缓冲液C的幻灯片在30-37℃下进行6小时或过夜，在湿润的腔室中。 注意:添加的LacZ的缓冲液C足够的容积;幻灯片不能孵化过程中干出来的。 </li><li>经过适当的孵育洗涤用PBS幻灯片补充了2mM的MgCl 2。冲洗部分简要双蒸2 O.盖玻片与Mayer的甘氨酸明胶。适合于光学显微镜配备有微分干涉对比（DIC）成像的设置。 </li></ol> 6. GENDER和示踪基因分型<ol><li>准备使用1M的Tris-HCl 1毫升pH为8.5，加入100μl的500mM EGTA尾裂解缓冲液，200微升10％SDS，和400微升的5M NaCl和DDH 2 O的以弥补为10ml的最终体积。 </li><li>加入500微升尾裂解缓冲液含有一个尾活检每个Eppendorf管。加入蛋白酶K至100毫克/毫升的最终浓度。孵育过夜在振荡器上在55℃。 </li><li>离心样品在17000×g离心10分钟，在RT。将上清液转移到新的离心管中。加入500微升异丙醇，以上清液。反相管上下混合至少5分钟。 </li><li>旋在离心机在17000×g离心5分钟，在室温。倒掉上清。加入200微升的70％乙醇。摇动至少5分钟。 </li><li>离心在17000×g离心5分钟。用的Pipetman（不要倒掉）上清。干燥温暖的室内（37°C）的颗粒为15-30分钟。 </li><li>在100微升悬浮颗粒使用大口径的过滤技巧的低TE pH值8.0或水。置于55℃1小时，并在37℃下过夜进行适当的溶解。保存在4℃。 </li><li>使用1微升的DNA进行PCR反应（含有1μl的尾DNA，2.5微升DMSO中，下午10点的每种引物，25μM的每种dNTP，和1M甜菜碱50μl的反应混合物）。 PCR引物基因分型和性别鉴定中列出的材料表。 </li></ol>

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No conflict of interest declared.

相比于立体定位注射示踪剂或neurotopic病毒的表达跨突触示踪剂作为转基因跟踪基因定义神经元群体的神经回路具有几个优点。首先，将示踪剂是作为内源蛋白质，因此不引起任何免疫反应和选择性的神经通路可在不同的动物具有较高的再现性进行分析。第二，由于这是一种非侵入性的方法也可用于从神经元用于立体定位注射不容易进入跟踪电路，例如在子宫内 。限制包括在跨突触传递一?...

解剖路径跟踪是最常用的工具，以破译大脑和行为1之间的关系之一。进步的神经回路追踪技术已经赋予与神经科学家从小鼠基因2确定神经元群追踪神经回路的功能。尽管这些技术上的进步仍然具有挑战性解开尤其是在胚胎成熟的神经回路的形成。这是因为大多数开发迄今跟踪方法是基于立体定位注射跨突触示踪剂的或遗传修饰的嗜神经病毒（ 图1）2,3。虽然这些技术实现空间和时间分辨率的连通，几个固有的局限性，如技术上具有挑战性示踪剂注射入发育中的大脑中，注射部位的再现性，潜在炎症在注射部位和最祁门功夫，功夫造成嗜神经病毒tantly毒性限制了其使用4。 另一种方法是要表达的跨突触示踪剂如遗传改造的小鼠转基因。我们最近修改了此技术，开发了二进制遗传跨突触跟踪系统，以地图的任何基因鉴定神经元群5的神经回路。我们的试验策略是基于两个新敲入小鼠品系，其表达或者双向示踪剂大麦凝集素（BL）的6或逆行示踪剂破伤风毒素片段C的ROSA 26轨迹融合到绿色荧光蛋白（GTT）7后的Cre介导的重组。在这里，我们用这些小鼠品系选择性表达BL和GTT的产生亲吻促动素的神经元，即牵连调节生殖轴8,9的成熟神经肽。我们表明，这种技术适用于可视吻的发育和成熟在雌性小鼠大脑5的胚胎发育过程中peptin神经电路。 育种策略在R26-BL-IRES-τlacZ（BIZ）和R26-GFP-TTC（GTT）示踪线敲入5株携带重组ROSA26等位基因。所述R26-BIZ和R26-GTT等位基因是转录沉默由于强烈的转录终止信号，它是由两个loxP序列侧翼5的存在。的BIZ和GTT转基因的表达是通过Cre重组酶介导的切除的转录终止信号的激活。在R26-BIZ和R26-GTT等位基因可以独立通过简单地用酶Cre车手穿越中使用。用于分析动物杂合的各自的Cre和R 26等位基因都可以使用。同窝承载1 的Cre或1 R 26等位基因，分别应用作对照。可替代地，也有可能产生吨riple敲入动物携带的Cre，R26-BIZ和R26-GTT等位基因，然而这将需要一个额外的十字架。

<table><tbody><tr><td>Bisbenzimide (Hoechst 33258 dye)</td><td>Sigma</td><td>14530-100MG</td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>32205-1L</td><td></td></tr><tr><td>Cryo mold (Peel-a-way)</td><td>Polyscience Inc.</td><td>18646A-1</td><td>22 x 22 x 20mm</td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418-100ML</td><td></td></tr><tr><td>Dimethyl Formamide (DMF)</td><td>VWR Chemicals</td><td>23470,293</td><td></td></tr><tr><td>EGTA</td><td>ROTH</td><td>3054.3</td><td></td></tr><tr><td>Fluoromount G</td><td>Southern Biotech</td><td>0100-01</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>G5882-50ML</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma</td><td>34988-7</td><td></td></tr><tr><td>Isopentane (Methyl 2-butane)</td><td>Sigma</td><td>M32631-2.5L</td><td></td></tr><tr><td>Kaiser&#039;s glycine gelatin</td><td>Merck</td><td>1092420100</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>494437-1L</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2670-100G</td><td></td></tr><tr><td>NaCl</td><td>ROTH</td><td>HN00.2</td><td></td></tr><tr><td>NBT</td><td>Sigma</td><td>298-83-9</td><td></td></tr><tr><td>Nonidet P40 substitute</td><td>Fluka</td><td>743.85</td><td></td></tr><tr><td>OCT</td><td>Leica</td><td>14020108926</td><td></td></tr><tr><td>PAP pen</td><td>Dako</td><td>S2002</td><td></td></tr><tr><td>Parafarmaldehyde</td><td>Sigma</td><td>P6148-1KG</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>Sigma</td><td>D6750-25G</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903-1KG</td><td></td></tr><tr><td>Superfrost slides</td><td>Thermo Scientific</td><td>FT4981GLPLUS</td><td></td></tr><tr><td>TSA kit</td><td>PerkinElmer</td><td>NEL700</td><td></td></tr><tr><td>TSA plus kit</td><td>PerkinElmer</td><td>NEL749A001KT</td><td></td></tr><tr><td>Tris</td><td>ROTH</td><td>AE15.2</td><td></td></tr><tr><td>Triton-X 100</td><td>ROTH</td><td>3051.2</td><td></td></tr><tr><td>Tween 20</td><td>ROTH</td><td>9127.1</td><td></td></tr><tr><td>X-gal</td><td>ROTH</td><td>2315.1</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>na</td><td></td></tr><tr><td>Light microscope equipped with DIC imaging</td><td>Zeiss</td><td>Axioskop2 equipped with Axio Vision software</td><td></td></tr><tr><td>Fluroscence microscope</td><td>Zeiss</td><td>Axioskop2 equipped with Axio Vision software</td><td></td></tr><tr><td>Photoshop</td><td>Adobe</td><td>PS6</td><td></td></tr><tr><td>Goat anti-WGA (recognizes BL)</td><td>Vector Laboatories</td><td>AS-2024</td><td></td></tr><tr><td>Biotinylayted horse anti-goat IgG</td><td>Vector Laboatories</td><td>BA-9500</td><td></td></tr><tr><td>Biotinylated goat anti-rabbit IgG</td><td>Vector Laboatories</td><td>BA-1000</td><td></td></tr><tr><td>Rabbit anti-GFP (recognizes GTT)</td><td>Invitrogen</td><td>A11122</td><td></td></tr><tr><td>Rabbit anti-GnRH</td><td>Affinity Bio Reagent</td><td>PA1-121</td><td></td></tr><tr><td>Dylight488-donkey anti-rabbit IgG</td><td>Thermo Scientific</td><td>SA5-10038</td><td></td></tr><tr><td>SA-Alexa Fluor 546</td><td>Life Technologies</td><td>S-11225</td><td></td></tr><tr><td colspan="4">Primers</td></tr><tr><td>BL Fwd (for BIZ genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>ATGAAGATGATGAGCACCAGGGC</td></tr><tr><td>BL Rev (for BIZ genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>AGCCCTCGCCGCAGAACTC</td></tr><tr><td>Cre Fwd (for Cre genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>GTCGATGCAACGAGTGATGAGGTTCG</td></tr><tr><td>Cre Rev (for Cre genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>CCAGGCTAAGTGCCTTCTCTACACCTGC</td></tr><tr><td>TTC Fwd (for GTT genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>AGCAAGGGCGAGGAGCTGTT</td></tr><tr><td>TTC Rev (for GTT genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>GTCTTGTAGTTGCCGTCGTCCTTGAA</td></tr><tr><td>XY Fwd (for gender genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>TGAAGCTTTTGGCTTTGA</td></tr><tr><td>XY Rev (for gender genotyping)</td><td>Eurofins MWG Operon</td><td></td><td>CCGCTGCCAAATTCTTTG</td></tr><tr><td>ROSA26 Fwd</td><td>Eurofins MWG Operon</td><td></td><td>CGAAGTCGCTCTGAGTTGTTATC</td></tr><tr><td>ROSA26 Rev</td><td>Eurofins MWG Operon</td><td></td><td>GCAGATGGAGCGGGAGAAAT</td></tr><tr><td>SA Rev</td><td>Eurofins MWG Operon</td><td></td><td>CGAAGTCGCTCTGAGTTGTTATC</td></tr></tbody></table>

conditional genetic transsynaptic tracing in the embryonic mouse brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

本节将展示可以得到与R26-BIZ（B L- 我 RES-τlacZ）和R26-GTT（G FP-TT C）等位基因工作代表性的结果。在这里，我们使用了R26-BIZ和R26-GTT等位基因来分析神经回路调节生殖轴的成熟。再生在脊椎动物的中心由神经元的下丘脑，其分泌的促性腺激素释放激素（GnRH）的一小部分进行控制。亲吻促动素，GnRH神经元的一个有力的活化剂，已被牵涉到调节GnRH神经元的活?...

Watch this Scientific Journal Video about Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain at JoVE.com

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

有条件的遗传跨突触标记在胚胎小鼠脑

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

conditional-genetic-transsynaptic-tracing-in-the-embryonic-mouse-brain

Research

JoVE Journal

Neuroscience

18.7K 次观看.  University of Saarland School of Medicine. Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution. 

视频: Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

科研

神经科学

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

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

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

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

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

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

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

激光制导神经跟踪在脑外植体

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用<em&gt;在Utero</em&gt;电穿孔

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

有条件的遗传跨突触标记在胚胎小鼠脑

摘要

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在子宫内电穿孔：鼠标控制时空基因Transefection

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

病毒遗传背景明确的神经回路跟踪

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

遗传操纵鼠标的发展下丘脑通过

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

双光子

激光制导神经跟踪在脑外植体

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

有条件的遗传跨突触标记在胚胎小鼠脑

摘要

探索更多视频

在子宫内电穿孔：鼠标控制时空基因Transefection

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘

病毒遗传背景明确的神经回路跟踪

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

遗传操纵鼠标的发展下丘脑通过

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

双光子

激光制导神经跟踪在脑外植体

延期共聚焦显像移植神经元在器官性切片培养胚胎小鼠脑使用

立体定位手术方法 ，通过 大水池在小鼠中显微注射尾部脑干和上颈椎脊髓

立体定位手术方法，通过大水池在小鼠中显微注射尾部脑干和上颈椎脊髓