我们感谢神户大学，日本的寺岛俊雄博士，教喉癌手术技术，以及普林斯顿大学的林恩·恩奎斯特供应PRV-巴萨博士。研究由美国国立卫生研究院先锋奖DP1 OD000448埃里希D.贾维斯和美国国家科学基金会研究生研究奖学金奖励给古斯塔沃·阿里亚加支持。从适当地记入以前的工作图是在公共科学图书馆ONE开放获取知识共享许可协议（CC-BY），根据该杂志的编辑方针使用。

注：所有动物的程序进行了审查和批准杜克大学实验动物管理及使用委员会。 1.存储伪狂犬病毒<ol><li>我们获得林恩·恩奎斯特博士在普林斯顿大学的实验室活病毒（PRV152）以1×10 9 PFU / m的效价。该协议产生的病毒已经出版2。 </li><li>分装病毒以每管20微升BSL-2生物安全柜和商店内于-80℃下适当的生物安全的条件。 </li><li>立即注射前解冻PRV的等分。 </li></ol> 2.手术准备注射到肌肉<ol><li>诱导通过注射氯胺酮 - 甲苯噻嗪的肌内全身麻醉（100mg / kg的氯胺酮; 10毫克/公斤赛拉嗪），并使用异氟烷维持适当的麻醉剂平面。 </li><li>保护角膜与眼用软膏。 </li><li>准备吨按照无菌技术通过修剪头发，用聚维酮碘交替磨砂和70％的酒精（至少3次）消毒手术部位，他的手术部位。请务必使用无菌单覆盖手术区域。请务必遵守无菌技术整个过程。 </li><li>做一个皮肤切口，揭示感兴趣的肌肉。例如，进入环甲喉肌，必须先删除胸骨舌骨肌上覆部分。 </li><li>任何密封切断肌肉VetBond组织粘合剂。 </li></ol> 3.注射伪狂犬病毒进入肌肉<ol><li>装载10微升NanoFil微量系统刚解冻的PRV的解决方案，附加一个34克的不锈钢针，并小心地将其安装在立体设备上。 </li><li>慢慢地清除死角和验证解决方案退出微量尖。丢弃液体作为bioharzard浪费。 </li><li>使用立体micropositioning设备的微量尖小心放置到感兴趣肌肉和慢慢填充肌肉直到轻微肿胀是可见的。喷射率将取决于肌肉和体积的大小而变化，以喷射。例如，200 NL（1微升数量），在4升/秒的速率五次喷射应1分钟间隔在同一地点，以填补环甲肌。移动注射器到感兴趣的肌肉下（在这种情况下，环杓侧），并重复注入过程。只有穿刺每一块肌肉一次。 </li><li>五分钟后缩回微量。 </li><li>密封在使用VetBond组织粘合剂筋膜断裂。 </li><li>毕竟注入已经完成，关闭使用VetBond组织粘合剂伤口。根据本地institional动物使用指南，缝合或伤口剪辑可以使用。 </li><li>监测动物，直到胸骨斜卧并提供镇痛，食物，水和护理要求由y我们的机构动物使用指南。 </li><li>后的实验确定生存时间（在这种情况下，90小时至标记2次皮层神经元），戊巴比妥过量牺牲动物和用0.9％盐水 ​​随后的4％甲醛的0.1M的PBS灌注transcardially。 </li><li>除去和后固定大脑中的4％甲醛24小时。 </li><li> Cryoprotect大脑中含30％蔗糖的至少48小时的磷酸缓冲液。 </li></ol> 4.免疫化学检测绿色荧光蛋白的<ol><li>切段在40微米的滑动切片，并保存浮动部分为0.1M PBS。较薄部分，例如30微米，可以染色安装部分时使用。 </li><li>淬火部分在0.3％的H 2 O 2的30分钟的PBS避光。 </li><li>方框中的部分的非特异性抗原在含有0.3％吐温20与来自VECTASTAIN精英套件正常山羊血清的PBS 30分钟（VE盒）。 </li><li&gt;孵育在兔网眼堵塞部分3.5小时抗eGFP的（1：1,000）在RT。 </li><li> 5分钟洗切片在PBS中三次，然后孵育它们1小时中的山羊抗兔二抗从VE试剂盒在RT。 </li><li>制备的ABC溶液从根据制造商的指示VE套件。 </li><li> 5分钟洗切片在PBS中三次，然后使它们反应1小时在ABC溶液从VE试剂盒在RT。 </li><li>在磷酸盐缓冲液进行10分钟清洗切片三次，并发展为在磷酸盐缓冲液8分钟，pH为7.4，含0.05％DAB（3，3&#39;-二氨基联苯胺）和0.015％的H 2 O 2。 </li><li>上Superfrost Plus或滑动安装部分和脱水它们通过一个分级醇系列（70％，95％，100％和100％，每次5分钟）。 </li><li>通过两个二甲苯洗涤（每次5分钟），清除部分和盖玻片Permount安装介质中的幻灯片。 </li><li>图像在显微镜安装的部分。民建联反应产物insidE中的细胞应该会出现褐色。数字化图像可以彩色倒要突出精细的工序。 </li></ol> 5.手术准备注射示踪成脑区通过PRV追踪发现<ol><li>诱导通过注射氯胺酮 - 甲苯噻嗪的肌内全身麻醉（100mg / kg的氯胺酮; 10毫克/公斤赛拉嗪），并使用异氟烷维持适当的麻醉剂平面。 </li><li>保护角膜与眼用软膏。 </li><li>修正头在适当的立体框架。 </li><li>根据无菌技术通过修整毛发和优碘交替磨砂和70％的醇（至少3个周期）消毒站点准备手术部位。 </li><li>使头皮切口并缩回皮在感兴趣的大脑区域。 </li><li>在适当的立体坐标进行一个小的开颅手术。例如，对于确定的PRV跟踪在一个laryngeally连接运动皮层的坐标ñ成年小鼠的1.2毫米横向0.2毫米喙前囟门。 </li></ol> 6.注射生物素葡聚糖胺进入脑<ol><li>通过将25毫克的北京经济技术开发区（1000万千瓦）的333微升无菌生理盐水的准备7.5％生物素葡聚糖胺（BDA）。 </li><li>加载Nanoject II微管系统为计划注入足够的BDA解决方案。 </li><li>慢慢地清除死角和验证的解决方案正在退出枪头。 </li><li>使用立体微定位装置小心地将枪头到感兴趣的脑区，并缓缓注入BDA。调整取决于区域的大小的喷射率来进行标记。例如，12注射4.6 NL应制成处于四个不同地点0.2毫米的间隔（rostro - 尾方向），以覆盖laryngeally连接运动皮层在成年小鼠。最后一次注射体积应根据所关注的大脑区域的大小来调整，牢记标签可以通过脑组织和沿标记的神经元的处理扩散从注射芯通过扩散。 </li><li>用牙科水泥密封了开颅手术，并关闭使用VetBond组织粘合剂头皮伤口。 </li><li>监测动物，直到胸骨斜卧并提供镇痛，食物，水和护理按要求通过体制动物使用指南。 </li></ol> 7.注射霍乱毒素B亚单位变成肌肉<ol><li>通过溶解在100μl的无菌盐水的1毫克CTB的制备1％霍乱毒素B亚基（CTB）。 </li><li> BDA注射入脑六天后，执行上述用于PRV注射到肌肉的手术准备。 </li><li>装载10微升NanoFil微量系统CTB的解决方案，附加一个34克的不锈钢针，并小心地将其安装在立体设备上。 </li><li>执行显微注射​​到感兴趣肌肉（多个）如前面解划为PRV。 </li><li>监测动物，直到胸骨斜卧并提供镇痛，食物，水和护理按要求通过体制动物使用指南。 </li><li> CTB注射后三天，通过戊巴比妥过量牺牲动物和灌注transcardially用0.9％盐水随后的4％甲醛的0.1M PBS中。 </li><li>除去和后固定大脑中的4％甲醛24小时。 </li><li> Cryoprotect大脑中含30％蔗糖的至少48小时的磷酸缓冲液。 </li></ol> 8.检测BDA和CTB在同一节<ol><li>切段在40微米的切片，并保存浮动部分为0.1M PBS。 </li><li>淬火部分在0.3％的H 2 O 2的30分钟的PBS避光。 </li><li>制备的ABC溶液从根据制造商的指示VE套件。 </li><li>在PBS中5分钟洗部分三次，然后使它们反应1小时在室温ABC溶液。 </LI&gt; <li>在磷酸盐缓冲液进行10分钟清洗切片三次，并发展为在磷酸盐缓冲液8分钟，pH为7.4，含0.05％DAB（3，3&#39;-二氨基联苯胺），0.015％的H 2 O 2，和0.05％的氯化镍。在细胞内的DAB反应产物应显示为黑色。 </li><li>块部分为在PBS中30分钟含有0.3％吐温20与来自VECTASTAIN精英套件正常兔血清。 </li><li>孵育在山羊网眼堵塞部分2小时抗CTB（1：10,000）于RT。 </li><li>在PBS中5分钟洗部分三次，然后孵育它们1小时在兔抗山羊二抗从VE试剂盒在RT。 </li><li>制备的ABC溶液从根据制造商的指示VE套件。 </li><li>在PBS中5分钟洗部分三次，然后使它们反应在在室温下的ABC溶液30分钟。 </li><li>洗部分在磷酸盐缓冲液三次10分钟，并发展为在磷酸盐缓冲液达8分钟，pH为7.4，含0.05％DAB（3,3&#39;二氨基联苯胺）和0.015％H 2 O 2。在细胞内的DAB反应产物应该出现褐色。 </li><li>上Superfrost Plus或滑动安装部分和脱水它们通过一个分级醇系列（70％，95％，100％和100％，每次5分钟）。 </li><li>通过两个二甲苯洗涤（每次5分钟），清除部分和盖玻片Permount安装介质中的幻灯片。 </li><li>图像在显微镜安装的部分。 </li></ol>

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroinvasiveness+of+pseudorabies+virus+injected+intracerebrally+is+dependent+on+viral+concentration+and+terminal+field+density.">Neuroinvasiveness of pseudorabies virus injected intracerebrally is dependent on viral concentration and terminal field density.</a> The Journal of Comparative Neurology. 407 (3), 438-452 (1999).</li><li>Pickard, G. E., Smeraski, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravitreal+injection+of+the+attenuated+pseudorabies+virus+PRV+Bartha+results+in+infection+of+the+hamster+suprachiasmatic+nucleus+only+by+retrograde+transsynaptic+transport+via+autonomic+circuits.">Intravitreal injection of the attenuated pseudorabies virus PRV Bartha results in infection of the hamster suprachiasmatic nucleus only by retrograde transsynaptic transport via autonomic circuits.</a> The Journal of Neuroscience. 22 (7), 2701-2710 (2002).</li><li>Smith, G. A., Gross, S. P., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+use+bidirectional+fast+axonal+transport+to+spread+in+sensory+neurons.">Herpesviruses use bidirectional fast axonal transport to spread in sensory neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (6), 3466-3470 (2001).</li></ol>

No conflicts of interest declared.

有一些必须规划使用PRV152 4,21的实验时，应考虑到的问题。最重要的是，伪狂犬病毒是致命的。正如前面提到的，大猿，包括人类不容易受到感染，但相应的必须小心保护其他动物。成年小鼠通常接种减毒PRV152应变后五至七天生存。因此，PRV152是不适合用于需要存活时间超过一周长的实验。此外，受感染的动物在生存期通常显示疾病的迹象，从而限制PRV152为需要观察的正常行为的实验工具...

跨突触跟踪已成为用于分析调节通过多突触电路周边靶中央传出神经的有力工具。这种方法已通过利用猪病原体伪狂犬病毒（PRV），尤其是减毒株PRV-巴萨在1961年首先描述最广泛使用的啮齿动物的大脑12。在这里，我们提出了一个协议，确定具体肌肉的运动皮层表现或使用重组伪狂犬病病毒株（PRV152）表达绿色荧光蛋白（EGFP）报告基因2的肌肉群。所描述的方法利用的嗜神经病毒，产生感染性子代跨突触感染功能电路3,4,13中的其他神经细胞的行为。 PRV152，这是同基因与PRV-巴萨，只有穿越突触逆行通过突触连接的分层顺序远离感染部位3,5- &lt;/ SUP&gt;。通过精确控制感染的周边部位也能够限制病毒的进入大脑通过运动神经元的特定子集。作为依次病毒感染连接神经元的链，eGFP的信号的整个大脑所得图案然后将解决在连接到最初感染的细胞的神经元网络。 使用病毒神经跟踪的一个附加的优点是报告蛋白（eGFP的在这种情况下）感染的细胞内的扩增。这个信号放大提供灵敏度的电平，它允许检测甚至稀疏突起。例如，一个稀疏突起从鼻毛运动皮层的面部运动神经元控制所述晶须被发现在使用病毒表达的绿色荧光蛋白14只大鼠 ;先前的研究没有使用经典的示踪剂没有报告基因扩增11,15找到这个投影。不幸的是，许多病毒追踪载体，如在引用的研究中使用的，不交叉的突触，从而限制了其用于跟踪多突触电路。 而呈现明显的优点，用于识别参与的电动机电路单元的网络，跨突触的分布式特性跟踪与PRV-152使得该电路难以解释内特定的连接。因此，我们提出了验证识别电路中的特定连接使用PRV-152双标使用生物素葡聚糖胺（BDA）和霍乱毒素B亚基（CTB）的简单方法。北京经济技术开发区和CTB的结合使用是一种行之有效的追踪神经元6-8,11的特定集合之间的连接方式。一起使用时，这两种示踪剂可以在相同的部分用双色DAB（3，3&#39;-二氨基联苯胺）过程16可视化。高分子量BDA（BDA10kDa）被选定为这个协议，因为它ÿields神经元突起6,7,9进行详细标注。 BDA10kDa的其它优点包括以下：它是在顺行方向6-8优先运输;它可以通过离子电渗或压力注射6-8交付;它可以通过简单的抗生物素蛋白-生物素化辣根过氧化物酶（ABC）的程序17被可视化;它可以通过光或电子显微镜6,7,18进行成像。免疫化学检测CTB的具有过氧化物和DAB被选择为运动神经元逆行标记，因为它是在揭示细胞过程，包括远侧树突10,19有效。我们最近使用这种方法来识别发声运动通路在小鼠和以显示从主运动皮层稀疏连接到喉运动神经元，这是以前假定为缺席20。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>NanoFil Microinjection System</td>
			<td>World Precision Instruments</td>
			<td>IO-Kit</td>
			<td>34 gauge option</td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>David Kopf Instruments</td>
			<td>Model 900</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II Auto-Nanoliter Injector</td>
			<td>Drummond Scientific Company</td>
			<td>3-000-204</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>VetBond</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane (Forane)</td>
			<td>Baxter&#160;</td>
			<td>1001936060</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine Swab Stick</td>
			<td>Cardinal Health</td>
			<td>2130-01</td>
			<td>200 count</td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated dextran amines</td>
			<td>Invitrogen</td>
			<td>D-1956</td>
			<td>10,000 MW</td>
		</tr>
		<tr>
			<td>Pseudorabies virus</td>
			<td>Laboratory of Dr. Lynn Enquist (Princeton University)</td>
			<td>PRV152</td>
			<td>Titer &#62; 1 x 107</td>
		</tr>
		<tr>
			<td>Anti-Cholera Toxin B Subunit (Goat)</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholera Toxin B Subunit</td>
			<td>List Biological Laboratories</td>
			<td>103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-eGFP</td>
			<td>Open Biosystems</td>
			<td>ABS4528</td>
			<td></td>
		</tr>
		<tr>
			<td>3, 3&#39;-diaminobenzidine</td>
			<td>Sigma-Aldrich</td>
			<td>D5905</td>
			<td>10 mg tablets</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td>Dilute to 4%</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>Sigma-Aldrich</td>
			<td>H3410</td>
			<td>30%</td>
		</tr>
		<tr>
			<td>Ketamine HCl &#38; Xylazine HCl</td>
			<td>Sigma-Aldrich</td>
			<td>K4138</td>
			<td>80 mg/mL &#38; 6 mg/mL</td>
		</tr>
		<tr>
			<td>Nickel chloride</td>
			<td>Sigma-Aldrich</td>
			<td>339350</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>P3619</td>
			<td>1.0 M; pH 7.4</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5493</td>
			<td>10X; pH 7.4</td>
		</tr>
		<tr>
			<td>Sodium Pentobarbital</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td>50 mg/mL dose</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>Histological grade</td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6101 (rabbit); PK-6105 (goat)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optixcare opthalmic ointment</td>
			<td>Vet Depot</td>
			<td>1017992</td>
			<td></td>
		</tr>
	</tbody>
</table>

transsynaptic tracing from peripheral targets with pseudorabies virus followed by cholera toxin and biotinylated dextran amines double labeling

跨突触跟踪已成为用于分析调节通过多突触电路周边靶中央传出神经的有力工具。这种方法已通过利用猪病原体伪狂犬病毒（PRV）1最广泛使用的大脑中的。 PRV不会感染大猿，包括人类，所以它是最常用的研究，小型哺乳动物，尤其是啮齿动物。的伪狂犬病应变PRV152表达增强的绿色荧光蛋白（EGFP）报告基因和只有逆行通过突触连接远离感染部位2,3的分层顺序跨过功能性突触。其他PRV毒株具有不同的微生物学性质，并且可以在两个方向（PRV-Becker和PRV-卡普兰）-4,5-运输。该协议将专门处理PRV152。通过提供该病毒在外围部位，如肌肉，也能够限制病毒的进入吨通过一组特定的神经元的他大脑。的eGFP信号的整个大脑所得图案然后解决了连接到最初感染的细胞的神经元。作为跨突触跟踪与伪狂犬病毒的分布特性使得解释中所标识的网络专用连接困难，我们目前采用生物素葡聚糖胺（BDA）和霍乱毒素B亚基（CTB）进行确认，使用鉴定细胞间的连接灵敏，可靠的方法PRV152。免疫化学检测BDA和CTB的具有过氧化物和DAB（3，3&#39;-二氨基联苯胺）的选择是因为它们是在揭示细胞过程，包括远侧树突6-11有效。

染色的绿色荧光蛋白应该开始呈现出微弱的信号在主运动神经元注入PRV152进入肌肉后大约72小时。的复制和病毒的跨突触传输是titer-和时间依赖性4。大约90小时注射后，绿色荧光蛋白染色将显示强劲的信号2次感染的细胞。较长的生存时间，就会发现3 次和更高阶的细胞，但生存时间由伪狂犬病病毒，在接种后5天左右的杀伤力有限。 图1a显示</stro...

Watch this Scientific Journal Video about Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling at JoVE.com

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 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 ...

transsynaptic-tracing-from-peripheral-targets-with-pseudorabies-virus

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Neuroscience

12.6K 次观看.  Duke University Medical Center. 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.

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

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

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

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 ...

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.

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 ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

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 ...

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of its labeling was affected by various factors, here, we provide a refined protocol for the application of high molecular weight BDA for studying optimal neural labeling in the central nervous system. After stereotactic injection of BDA into the ventral posteromedial nucleus (VPM) of the thalamus in the rat through a delicate glass pipette, BDA was stained with fluorescent streptavidin-Alexa (AF) 594 and counterstained with fluorescent Nissl stain AF500/525. On the background of green Nissl staining, the red BDA labeling, including neuronal cell bodies and axonal terminals, was more distinctly demonstrated in the somatosensory cortex. Furthermore, double fluorescent staining for BDA and the calcium-binding protein parvalbumin (PV) was carried out to observe the correlation of BDA labeling and PV-positive interneurons in the cortical target, providing the opportunity to study the local neural circuits and their chemical characteristics. Thus, this refined method is not only suitable for visualizing high quality neural labeling with the high molecular weight BDA through reciprocal neural pathways between the thalamus and cerebral cortex, but also will permit the simultaneous demonstration of other neural markers with fluorescent histochemistry or immunochemistry.

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

高分子量生物素化葡聚糖胺在大鼠丘脑-皮质投影示踪中的应用

High molecular weight biotinylated dextran amine (BDA) has been used as a highly sensitive neuroanatomical tracer for many decades. Since the quality of ...

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 ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...