我们要感谢药用处方。 Huibert Mansvelder和Bert索​​克曼的广泛支持，马塞尔Oberlaender的富有成果的讨论，并提供技术援助，提供神经元追踪，和Brendan Lodder博士。使用ntrode VI用于LabVIEW，由R.布鲁诺（哥伦比亚大学。，NY，USA）慷慨地提供了数据被收购。这项研究是由马克斯·普朗克学会和伯恩斯坦计算神经科学中心，图宾根的支持（由德国联邦教育与研究部（BMBF资助; FKZ:01GQ1002））（RTN），中心Neurogenomics与认知研究（CNCR） ，神经科学校园阿姆斯特丹（NCA），资金CPJdK（NWO-ALW＃822.02.013和ENC-网络＃P3-C3）和阿姆斯特丹VU大学。

1。动物的制备所有实验步骤都进行了按照荷兰法律和评估后，由当地的伦理委员会，在阿姆斯特丹VU大学，荷兰。 <ol><li>麻醉Wistar系大鼠（P25-P45，♂/♀）用异氟烷（在氧气2-3％），随后与氨基甲酸乙酯（20％在0.9％NaCl，1.6〜1.7克/公斤）通过腹膜内注射。通过监测捏撤出，眼睑反射消失，并且触须运动评估麻醉深度。 </li><li>的情况下施用额外剂量的尿烷（初始剂量的10％）的动物未达到足够的麻醉深度或每当触须抽搐都在实验过程中观察到的。 </li><li>将麻醉的动物的立体框架装有加热垫，插入直肠温度探头和通过加热垫保持动物的体温在37.5±0.5°C。 </li><l我&gt;放置动物的头在立体框架，用剪刀来呈现皮肤可见修剪头顶头发。 </li><li>注入100微升1％利多卡因（在0.9％NaCl）皮下注射局部麻醉在操作现场，等待3-5分钟。通过切割在尾至头端方向去除皮肤覆盖颅骨的表面和清洁剩余的组织。 </li><li>用0.9％NaCl和吸水拖把打扫广泛的头骨。 </li><li>确定目标区域的左侧半球（初级躯体感觉皮层（S1）:2.5毫米后5.5 mm处至前囟门）的坐标。与手术皮肤记号笔标记头骨上的位置。 </li><li>刮去骨轻轻地从感兴趣区域牙钻，直到骨变得透明和血管清晰可见。 </li><li>作为0.5mm×0.5毫米开颅通过切割在减薄的颅骨窗用手术刀（＃11），以避免损坏硬膜和血管。可选:采用外科皮肤记号笔提高知名度纪念开颅手术的边缘。 </li><li>取适量涂抹于干燥的头骨，然后牙科水泥薄薄的一层强力胶来构造一个浴（ 即录音室）封闭的开颅手术。 </li><li>修剪胡须在对侧到正好是5毫米的晶须卵泡。可选:突出修剪胡须与黑色睫毛膏。 </li></ol> 2。 Juxtasomal录音和生物胞素标记<ol><li>使硼硅玻璃的补丁移液器〜1微米的内顶圆直径，以获得电极用3-5MΩ电阻。注意:理想移液器形态为juxtasomal记录是一个渐进的细长锥形，低锥角和〜1μm的末端（ 图1）。 </li><li>根据记录的深度（相对于软脑膜表面）时，记录电极的锥形尺寸应调整。极为重要的是，吨他锥度的直径应小于75微米的点在大脑中的条目以避免机械损伤或应力的记录区域。这表明，对于浅表皮层录音，300-500微米带的最大75微米的外径的锥度是必需的（ 图1A），而记录从相对较深的皮质层要求的1,500-1,800微米的较长的锥度，又具有最大外直径为75μm（在1,800μm的从电极头）。 </li><li>加载补丁吸管与正常大鼠铃声（NRR）添加2％生物胞素（ 见表1解决方案和试剂），并安装补丁吸管的头阶段固定在显微操作。 </li><li>电极架的角度设置为34°相对于矢状平面专门针对大鼠S1的D2柱。 </li><li>头段连接到网桥模式的放大器（ 即电流钳）。 </li><li>定位补丁吸取邻近的开颅手术。填补浴用0.9％NaCl和确定电极的电阻应是3-5兆欧，通过施加矩形脉冲​​具有正电流注入（1 nA的，200毫秒开/关）之间分摊。 </li><li>建立100-150毫巴超压，推进补丁吸管在1微米的步骤，同时施加正电流的方波脉冲（1 nA的，200毫秒开/关）。监视健壮的电阻变化时建立与硬脑膜接触。在这一点上，设置微操作的坐标为"零"，以允许准确的深度测量。 </li><li>推进1微米步模式，直到补丁吸管穿透由电极电阻突然下降显示硬膜。取出吸移管的夹持压力。 </li><li>搜索单个单位，而在1微米的步骤前进。通过施加200毫秒的通/断脉冲持续监测电极电阻。在电极电阻表型增长盟友表示单个神经元的接近。推进电极，直到正动作电位（AP）的〜2毫伏的波形记录（ 图2A和2B）。 </li><li>可选:记录神经元的自发扣球图案和确定晶须诱发扣球通过，例如，尾侧偏转为200毫秒个别晶须以3.3°的角度使用压电装置18。 </li><li>推进电极，直到阻力25-35MΩ和尖峰有3-8 mV的幅度得到juxtasomal灌装的最佳条件。通过施加矩形脉冲​​的正电流（nA的1，200毫秒的开启/关闭）开始juxtasomal填充。同时监测AP的波形和频率（ 图2A和2B）慢慢地，逐渐增加电流步长为0.1 nA的。 </li><li>监测过程中的导通相位的块脉冲（ 图2C的膜孔中的AP的频率明显增加</STRONG&gt;）。加注期间的尖峰波形示出了增加的宽度和减小后超极化（ 图2C和2D）。其他参数包括增加噪音或小（1-5毫伏）负直流偏移。 </li><li>增加或减少电流（1 NA）却使以维持稳定的生物胞素滴注。减少或根据该AP的频率突然增加停止的电流脉冲，以避免毒性通过细胞外离子，如钠离子过量涌入。注意:每个神经元会向电流注入和juxtasomal标记参数需要根据个别拍摄条件进行调整，有不同的反应。 </li><li>停止当前注射后密切监测信号。经过灌装届穗波形通常是变宽，呈超极化后大大减少。等待的神经元，这是明显的，当尖峰波形返回到其原有的特性（ 即存在的恢复正常的后超极化， 图2）。 </li><li>重复生物胞素神经元的完全恢复后充填阶段，以增加生物胞素负荷，以提高染色质量。 </li><li>收回补丁吸管，步长为1微米，直到尖峰幅度减小，以减少任何机械应力的单元格。等待至少1小时的生物胞素弥漫在细胞内，同时密切监测动物的体温和呼吸。 </li></ol> 3。灌注动物及取出脑<ol><li>准备灌注安装，冲洗，并预装有0.9％的NaCl油管。 </li><li>放置动物在手术托盘和标准标签胶带将其固定。确保麻醉足够的深度，脚捏和眼睑反射应该缺席。 </li><li>使内侧穿过腹壁只是肋骨下方到外侧切口（5-6厘米），并继续在后前方向，露出胸骨。向前拉胸骨，仔细从隔膜分离肝脏。 </li><li>作一个小切口隔膜，通过下部肋骨切开并继续沿腹腔暴露心脏的整个长度的切口。 </li><li>取出心包。 </li><li>将针头插入左心室，并在右心房切口。灌注用0.9％的NaCl（约8毫升/分钟），直到肝脱色完成。 </li><li>输液切换到4％多聚甲醛（PFA），直到前爪的刚度和下颚是显而易见的。 </li><li>用剪刀斩首鼠</li><li>除去剩余的颈部肌肉完全暴露颅骨。 </li><li>定位在脑干的剪刀上的背侧并小心切骨沿矢状缝，保持背侧的位置。 </li><li>从矢状缝两侧取出骨头使用镊子，露出大脑。小心取出硬脑膜避免Ð豪悦国际。 </li><li>小心地将钝铲到大脑的腹侧，轻轻取出大脑。 </li><li>后修复整个大脑过夜PFA在4°C大脑切换到0.05M磷酸盐缓冲液（PB）和储存于4℃。 </li></ol> 4。切片大脑中切段<ol><li>就拿大脑出了0.05M的PB，并把它放在眼前面临的滤纸。用锋利的剃刀刀片来切断小脑沿冠状面和沿中间矢状面切割分离半球。 </li><li>应用强力胶安装平台上安装左半球其与前朝右矢状面。固定安装平台45°的vibratome角和淹没大脑在0.05M PB。 </li><li>固定在vibratome刀片，并确保在第一与脑表面接触是在半球体的前 - 后平面的中间。削减24 100微米节，并收集他们在含有0.05M PB 24孔板。 </li></ol> 5。组织程序<ol><li>根据先前描述的方法25，用于细胞色素氧化酶染色24和 ​​抗生物素蛋白-生物素-过氧化物酶方法进行组织学协议。可选:可视化生物胞素使用荧光亲和素/链霉亲和Alexa的结合物。这还允许双重染色逆行或顺行追踪技术。 </li><li>洗部5×5分钟用0.05M PB和准备3，3&#39;-二氨基联苯胺四盐酸盐（DAB）含有用于细胞色素氧化酶染色来可视化桶层4的S1（L4）的溶液（ 见表2溶液和试剂）。在孵育30-45分钟的预热的溶液部分6-12由软脑膜，于37℃下</li><li>冲洗切片用0.05M PB为6×5分钟和孵育所有部分在3％H 2解渴内源性过氧化物酶的活性</子&gt; O 2在0.05M PB为20分钟，在室温（RT）。 </li><li>冲洗切片用0.05M PB为5×10分钟。在孵育ABC-解决方案的部分（ 见表3解决方案和试剂）一夜之间在4°C。 </li><li>冲洗切片用0.05M PB为5×10分钟，并准备民建联溶液以可视化的生物胞素填充神经元（ 见表4解决方案和试剂）。在孵育过滤后的溶液部分在室温为45-60分钟。 </li><li>冲洗切片用0.05M PB为5×10分钟。山路段在显微镜玻片和盖玻片用的Mowiol（ 见表5解决方案和试剂）。 </li><li>确定使用光学显微镜的标签质量。 </li></ol>

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U.S.A. 106 (38), 16446-16450 (2009).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Horikawa, K., Armstrong, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+means+of+intracellular+labeling:+injection+of+biocytin+and+its+detection+with+avidin+conjugates.">A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates.</a> J. Neurosci. Methods. 25 (1), 1-11 (1988).</li><li>O'Connor, D. H., Peron, S. 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S., 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=Semi-automated+three-dimensional+reconstructions+of+individual+neurons+reveal+cell+type-specific+circuits+in+cortex.">Semi-automated three-dimensional reconstructions of individual neurons reveal cell type-specific circuits in cortex.</a> Commun. Integr. Biol. 4 (4), 486-488 (2011).</li><li>Bruno, R. M., Hahn, T. T., Wallace, D. J., de Kock, C. P., Sakmann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+experience+alters+specific+branches+of+individual+corticocortical+axons+during+development.">Sensory experience alters specific branches of individual corticocortical axons during development.</a> J. Neurosci. 29 (10), 3172-3181 (2009).</li><li>Schubert, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+without+disturbing:+how+different+cortical+neuron+classes+represent+tactile+stimuli.">Observing without disturbing: how different cortical neuron classes represent tactile stimuli.</a> J. Physiol. 581 (1), 5 (2007).</li><li>Neumann, E., Kakorin, S., Toensing, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamentals+of+electroporative+delivery+of+drugs+and+genes).">Fundamentals of electroporative delivery of drugs and genes).</a> Bioelectrochem. Bioenerg. 48 (1), 3-16 (1999).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. 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该authros什么都没有透露。

该juxtasomal方法允许记录在体内动作电位扣球来自全国各地的行为条件的单台（麻醉，清醒头固定或自由活动）与生物胞素标记的神经元记录为事后的细胞类型分类和/或三维重建的选项。主要的优点是能够获得在细胞外（因此非侵入性）构型的生理参数，还能够与生物胞素16,17,32细胞内标记的神经元。除了 ​​生物胞素标记，这种技术可以用于注射神经元与DNA，RNA，蛋白质，...

神经网络由多种细胞类型，其特点是高度特异性的形态和生理特性1-7。因此，个别细胞类型的网络内执行专门任务的（参见例如Gentet 等人 8和Burgalossi 等 9）。我们才刚刚开始了解整个神经网络的细胞类型特异性的功能现在还是有很多被发现。为此，许多实验室正在实施的实验方法，使从生理参数已取得1,10-15相同的神经元群的形态特征进行分析。在这里，我们展示了juxtasomal标记技术16,17涉及与所记录的神经元与生物胞素的电结合使用传统的补丁移液器在细胞外（因此无创）配置电生理记录。该这种方法的主要优点是，非侵入性的性质确保了单个神经元的动作电位扣球被记录而不改变（ 如透析）的细胞的细胞内含量。其次是电，在juxtasomal方法提供事后细胞识别和重建的选项链接功能（生理），以结构（形态）。通常情况下，形态重构涉及重建的树突和轴突的形态，可以延长至脊柱和/或布顿密度的定量，甚至在用电子显微镜纳米级分辨率重建神经元形态的。该juxtasomal记录技术可以用于各种细胞类型跨越皮质层或子皮质区的范围内的物种的体内录音，虽然大多数研究已应用于该技术在小型啮齿动物如小鼠或大鼠。我们的研究重点是记录和标记的神经元从大鼠初级躯体感觉皮层（S1）和涉及视觉识别记录的神经元18，与精确定位在一个标准化的参考帧组合树突重建逆向工程皮质网络4,19和轴突结构的详细的重建来表征细胞类型特异性地方和远距离投射目标20。 相比其它体内记录技术（细胞内或细胞的全细胞），juxtasomal录音是相对稳定的，因此可以被跨越行为状态应用包括麻醉21,22，镇静剂14，醒头固定23，或什至可自由移动的动物9 。这里，我们表明在聚氨酯麻醉大鼠S1 juxtasomal标签，虽然我们强调该技术的普遍适用性来选择的许多制剂。

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juxtasomal biocytin labeling to study the structure-function relationship of individual cortical neurons

大脑皮质的特征是多层次和许多不同的细胞类型，它们共同作为网络负责许多高级认知功能，包括决策，感官引导行为或内存。为了理解如何这样复杂的神经元网络执行这样的任务，一个关键的第一步是确定在网络中的单个细胞类型的函数（或电活性），优选当动物正在执行一个相关的认知任务。另外，为了确定单个神经元网络和形态学的架构，以允许反向工程皮质网络的解剖结构是同样重要的。可今天的技术突破允许录制清醒细胞活性，动物行为与事后识别记录神经元的有价值的选择。在这里，我们展示了juxtasomal生物胞素标记技术，它涉及到记录动作电位人在细胞外（或宽松的补丁）配置扣球使用传统的补丁移液器。该juxtasomal记录配置是横跨行为条件相对稳定且适用，包括麻醉，镇静，清醒头固定，并且即使在可自由移动的动物。因此，这种方法允许在动物的行为来重建单个神经元，最终，整个皮质微电路的连接细胞类型特异性动作电位扣球。在这个视频稿件，我们将展示如何在juxtasomal配置单个神经元可在氨基甲酸乙酯麻醉大鼠的事后鉴定和形态重构被贴上生物胞素。

详细的知识对单个神经元的三维结构对于阐明神经元网络的组织原则至关重要。我们的方法包括一个管道，以实现高品质的生物胞素标记从体内制备，从而使事后神经元分类和树突和单个神经元，在高分辨率的轴突架构的详细重建。根据juxtasomal标签的质量，神经元恢复与不同的DAB-强度，从微弱到可以非常精确地对应于记录位置19,26位置激烈的DAB信号。在我们的实验室，一个?...

Watch this Scientific Journal Video about Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons at JoVE.com

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

要了解神经网络的结构，单个神经元的功能和形态特征是必要的。在这里，我们证明juxtasomal生物胞素标记，其允许电生理记录在胞外结构，但保持对胞内标记的神经元为事后重建的树突和轴突体系结构的能力。

Juxtasomal生物胞素标记来研究单个皮层神经元的结构与功能的关系

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

juxtasomal-biocytin-labeling-to-study-structure-function-relationship

Research

JoVE Journal

Neuroscience

13.5K 次观看.  VU University Amsterdam. 要了解神经网络的结构，单个神经元的功能和形态特征是必要的。在这里，我们证明juxtasomal生物胞素标记，其允许电生理记录在胞外结构，但保持对胞内标记的神经元为事后重建的树突和轴突体系结构的能力。 

视频: Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

激光捕获显微切割技术的果蝇外周神经元

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

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 ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

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 ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

Juxtasomal生物胞素标记来研究单个皮层神经元的结构与功能的关系

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