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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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><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 G 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 colspan="4">[header]</td></tr><tr><td>VetBond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Isofluorane (Forane)</td><td>Baxter&amp;nbsp;</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 &gt;1 x 10&lt;sup&gt;7&lt;/sup&gt;</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&#039;-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 &amp;amp; Xylazine HCl</td><td>Sigma-Aldrich</td><td>K4138</td><td>80 mg/ml &amp;amp; 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

Universidad de Cartagena UDEC

Research

JoVE Journal

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

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 ...

科研

神经科学

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 ...

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 ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

同时双光子<em&gt;在体内</em&gt;突触输入的影像和突触后目标中的鼠标Retrosplenial的Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

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 ...

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

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

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

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

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

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. Traditional in vitro electrophysiological techniques use electrical stimulation to evoke synaptic transmission. A major drawback of this method is its nonspecific nature; all axons in the region of the stimulating electrode will be activated, making it difficult to attribute an effect to a particular afferent connection. This issue can be overcome by replacing electrical stimulation with optogenetic-based stimulation. We describe a method for combining optogenetics with in vitro patch-clamp recordings. This is a powerful tool for the study of both basal synaptic transmission and synaptic plasticity of precise anatomically defined synaptic connections and is applicable to almost any pathway in the brain. Here, we describe the preparation and handling of a viral vector encoding channelrhodopsin protein for surgical injection into a pre-synaptic region of interest (medial prefrontal cortex) in the rodent brain and making of acute slices of downstream target regions (lateral entorhinal cortex). A detailed procedure for combining patch-clamp recordings with synaptic activation by light stimulation to study short- and long-term synaptic plasticity is also presented. We discuss examples of experiments that achieve pathway- and cell-specificity by combining optogenetics and Cre-dependent cell labeling. Finally, histological confirmation of the pre-synaptic region of interest is described along with biocytin labeling of the post-synaptic cell, to allow further identification of the precise location and cell type.

Ex Vivo Optogenetic Interrogation of Long-Range Synaptic Transmission and Plasticity from Medial Prefrontal Cortex to Lateral Entorhinal Cortex

Here we present a protocol describing viral transduction of discrete brain regions with optogenetic constructs to permit synapse-specific electrophysiological characterization in acute rodent brain slices.

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

Studying the physiological properties of specific synapses in the brain, and how they undergo plastic changes, is a key challenge in modern neuroscience. ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

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

摘要

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此视频中的章节

病毒遗传背景明确的神经回路跟踪

有条件的遗传跨突触标记在胚胎小鼠脑

激光制导神经跟踪在脑外植体

同时双光子

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

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

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

扩展轴突转运体内成像工具包

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

摘要

探索更多视频

此视频中的章节

病毒遗传背景明确的神经回路跟踪

有条件的遗传跨突触标记在胚胎小鼠脑

激光制导神经跟踪在脑外植体

同时双光子

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

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

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

在河马区文特尔区谷氨酸终端的 Vivo 解剖和功能跟踪组合

离体 从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问

扩展轴突转运体内成像工具包

离体从内侧前额叶皮层到外嗅皮层的远距离突触传递和可塑性的光遗传学询问