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 border="0" cellpadding="0" cellspacing="0" width="721">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:219px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bisbenzimide (Hoechst 33258 dye)</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">14530-100MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">32205-1L</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryo mold (Peel-a-way)</td>
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			<td style="width:119px;">18646A-1</td>
			<td>22mm x 22mm x 20mm</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">D8418-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Formamide (DMF)</td>
			<td style="width:219px;">VWR Chemicals</td>
			<td style="width:119px;">23470,293</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EGTA</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">3054.3</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fluoromount G</td>
			<td style="width:219px;">Southern Biotech</td>
			<td style="width:119px;">0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">G5882-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">34988-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopentane (Methyl 2-butane)</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">M32631-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kaiser&#39;s Glycine gelatin</td>
			<td style="width:219px;">Merck</td>
			<td style="width:119px;">1092420100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">494437-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:219px;">Sigma</td>
			<td>M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">HN00.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NBT</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">298-83-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nonidet P40 substitute</td>
			<td style="width:219px;">Fluka</td>
			<td style="width:119px;">743.85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">OCT</td>
			<td style="width:219px;">Leica</td>
			<td style="width:119px;">14020108926</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PAP pen</td>
			<td style="width:219px;">Dako</td>
			<td style="width:119px;">S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafarmaldehyde</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">P6148-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium deoxycholate</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">D6750-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:219px;">Sigma</td>
			<td style="width:119px;">S7903-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Superfrost slides</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">FT4981GLPLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TSA kit</td>
			<td style="width:219px;">PerkinElmer&#160;</td>
			<td style="width:119px;">NEL700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TSA plus kit</td>
			<td style="width:219px;">PerkinElmer&#160;</td>
			<td style="width:119px;">NEL749A001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">AE15.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton-X 100</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">3051.2</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween 20</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">9127.1</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-gal</td>
			<td style="width:219px;">ROTH</td>
			<td style="width:119px;">2315.1</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryostat</td>
			<td style="width:219px;">Leica</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Light microscope equipped with DIC imaging&#160;</td>
			<td style="width:219px;">Zeiss</td>
			<td style="width:119px;">Axioskop2 equipped with Axio Vision software</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fluroscence microscope</td>
			<td style="width:219px;">Zeiss</td>
			<td style="width:119px;">Axioskop2 equipped with Axio Vision software</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Photoshop</td>
			<td style="width:219px;">Adobe</td>
			<td style="width:119px;">PS6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Goat anti-WGA (recognizes BL)</td>
			<td style="width:219px;">Vector Laboatories</td>
			<td style="width:119px;">AS-2024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylayted horse anti-goat IgG</td>
			<td style="width:219px;">Vector Laboatories</td>
			<td style="width:119px;">BA-9500&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated goat anti-rabbit IgG</td>
			<td style="width:219px;">Vector Laboatories</td>
			<td style="width:119px;">BA-1000&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rabbit anti-GFP (recognizes GTT)</td>
			<td style="width:219px;">Invitrogen</td>
			<td style="width:119px;">A11122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit anti-GnRH</td>
			<td style="width:219px;">Affinity Bio Reagent</td>
			<td style="width:119px;">PA1-121</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dylight488-donkey anti-rabbit IgG</td>
			<td style="width:219px;">Thermo Scientific</td>
			<td style="width:119px;">SA5-10038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SA-Alexa Fluor 546</td>
			<td style="width:219px;">Life Technologies</td>
			<td>S-11225</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primers</td>
			<td style="width:219px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL Fwd (for BIZ genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">&#160;ATGAAGATGATGAGCACCAG 
			GGC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL Rev&#160; (for BIZ genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">&#160;AGCCCTCGCCGCAGAACTC&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cre Fwd&#160; (for Cre genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">GTCGATGCAACGAGTGATGAG 
			GTTCG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cre Rev&#160; (for Cre genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">CCAGGCTAAGTGCCTTCTCTAC 
			ACCTGC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TTC Fwd&#160; (for GTT genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">AGCAAGGGCGAGGAGCTGTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TTC Rev&#160; (for GTT genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">GTCTTGTAGTTGCCGTCGTCCT 
			TGAA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XY Fwd (for gender genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">TGAAGCTTTTGGCTTTGA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XY Rev&#160; (for gender genotyping)</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">CCGCTGCCAAATTCTTTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ROSA26 Fwd</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2">CGAAGTCGCTCTGAGTTGTTATC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">ROSA26 Rev</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2" style="width:285px;">GCAGATGGAGCGGGAGAAAT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SA Rev</td>
			<td>Eurofins MWG Operon</td>
			<td colspan="2" style="width:285px;">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

18.6K 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

Manual Drainage of the Zebrafish Embryonic Brain Ventricles

Cerebrospinal fluid (CSF) is a protein rich fluid contained within the brain ventricles. It is present during early vertebrate embryonic development and persists throughout life. Adult CSF is thought to cushion the brain, remove waste, and carry secreted molecules1,2. In the adult and older embryo, the majority of CSF is made by the choroid plexus, a series of highly vascularized secretory regions located adjacent to the brain ventricles3-5. In zebrafish, the choroid plexus is fully formed at 144 hours post fertilization (hpf)6. Prior to this, in both zebrafish and other vertebrate embryos including mouse, a significant amount of embryonic CSF (eCSF) is present . These data and studies in chick suggest that the neuroepithelium is secretory early in development and may be the major source of eCSF prior to choroid plexus development7.
eCSF contains about three times more protein than adult CSF, suggesting that it may have an important role during development8,9. Studies in chick and mouse demonstrate that secreted factors in the eCSF, fluid pressure, or a combination of these, are important for neurogenesis, gene expression, cell proliferation, and cell survival in the neuroepithelium10-20. Proteomic analyses of human, rat, mouse, and chick eCSF have identified many proteins that may be necessary for CSF function. These include extracellular matrix components, apolipoproteins, osmotic pressure regulating proteins, and proteins involved in cell death and proliferation21-24. However, the complex functions of the eCSF are largely unknown.
We have developed a method for removing eCSF from zebrafish brain ventricles, thus allowing for identification of eCSF components and for analysis of the eCSF requirement during development. Although more eCSF can be collected from other vertebrate systems with larger embryos, eCSF can be collected from the earliest stages of zebrafish development, and under genetic or environmental conditions that lead to abnormal brain ventricle volume or morphology. Removal and collection of eCSF allows for mass spectrometric analysis, investigation of eCSF function, and reintroduction of select factors into the ventricles to assay their function. Thus the accessibility of the early zebrafish embryo allows for detailed analysis of eCSF function during development.

We present a method to collect cerebrospinal fluid (CSF) and to create a system which lacks CSF within the embryonic zebrafish brain ventricular system. This allows for further examination of CSF composition and its requirement during embryonic brain development.

手动排水的斑马鱼胚胎脑室

Cerebrospinal fluid  (CSF) is a protein rich fluid contained within the brain ventricles. It is  present during early vertebrate embryonic development and ...

科研

神经科学

Selective Tracing of Auditory Fibers in the Avian Embryonic Vestibulocochlear Nerve

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling of auditory axons within the VIIIth cranial nerve would enhance the study of central auditory circuit development. This approach is challenging because multiple sensory organs of the inner ear contribute to the VIIIth nerve 1. Moreover, markers that reliably distinguish auditory versus vestibular groups of axons within the avian VIIIth nerve have yet to be identified. Auditory and vestibular pathways cannot be distinguished functionally in early embryos, as sensory-evoked responses are not present before the circuits are formed. Centrally projecting VIIIth nerve axons have been traced in some studies, but auditory axon labeling was accompanied by labeling from other VIIIth nerve components 2,3. Here, we describe a method for anterograde tracing from the acoustic ganglion to selectively label auditory axons within the developing VIIIth nerve. First, after partial dissection of the anterior cephalic region of an 8-day chick embryo immersed in oxygenated artificial cerebrospinal fluid, the cochlear duct is identified by anatomical landmarks. Next, a fine pulled glass micropipette is positioned to inject a small amount of rhodamine dextran amine into the duct and adjacent deep region where the acoustic ganglion cells are located. Within thirty minutes following the injection, auditory axons are traced centrally into the hindbrain and can later be visualized following histologic preparation. This method provides a useful tool for developmental studies of peripheral to central auditory circuit formation.

Here we describe a microdissection technique followed by fluorescent dye injection into the acoustic ganglion of early chick embryos for selective tracing of auditory axon fibers in the nerve and hindbrain.

禽流感胚胎听神经听觉神经纤维的选择性跟踪

The embryonic chick is a widely used model for the study of peripheral and central ganglion cell projections. In the auditory system, selective labeling ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

从外围目标跨突触跟踪与伪狂犬病病毒其次是霍乱毒素和生物素葡聚糖胺双标

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 ...

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 ...