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. P., Huber, D., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+in+barrel+cortex+underlying+vibrissa-based+object+localization+in+mice.">Neural activity in barrel cortex underlying vibrissa-based object localization in mice.</a> Neuron. 67 (6), 1048-1061 (2010).</li><li>Veinante, P., Deschenes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+study+of+motor+cortex+projections+to+the+barrel+field+in+rats.">Single-cell study of motor cortex projections to the barrel field in rats.</a> J. Comp. Neurol. 464 (1), 98-103 (2003).</li><li>Boudewijns, Z. 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=Layer-specific+high-frequency+action+potential+spiking+in+the+prefrontal+cortex+of+awake+rats.">Layer-specific high-frequency action potential spiking in the prefrontal cortex of awake rats.</a> Front. Cell. Neurosci. 7, 99 (2013).</li><li>Oberlaender, M., Bruno, R. M., Sakmann, B., Broser, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitted+light+brightfield+mosaic+microscopy+for+three-dimensional+tracing+of+single+neuron+morphology.">Transmitted light brightfield mosaic microscopy for three-dimensional tracing of single neuron morphology.</a> J. Biomed. Opt. 12 (6), 064029 (2007).</li><li>Boudewijns, Z. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29 (3), 583-591 (2001).</li></ol>

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。

<table><tbody><tr><td>SM-6 control system</td><td>Luigs Neumann</td><td></td><td></td></tr><tr><td>LN- Mini 23 XYZ</td><td></td><td></td><td></td></tr><tr><td>LN- Mini 55 Manipulatorblock X2</td><td></td><td></td><td></td></tr><tr><td>Lynx-8 amplifier</td><td>Neuralynx</td><td></td><td></td></tr><tr><td>Axoclamp-2B amplifier</td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Osada model EXL-M40</td><td>Osada, Inc.</td><td></td><td></td></tr><tr><td>Piezoelectric device</td><td>Physik Instrumente</td><td>PL140.10</td><td></td></tr><tr><td>LabView</td><td>National Instruments, Austin, TX, USA</td><td></td><td>LabView acquisition software</td></tr><tr><td>Ntrode Virtual Instrument&amp;nbsp;</td><td>R. Bruno, Columbia Univ., NY</td><td></td><td></td></tr><tr><td>Sugi absorbent swabs</td><td>Kettenbach</td><td>30601</td><td></td></tr><tr><td>Cytochrome C from equine heart</td><td>Sigma</td><td>C2506</td><td></td></tr><tr><td>Catalase from bovine liver</td><td>Sigma</td><td>C9322</td><td></td></tr><tr><td>DAB</td><td>Sigma</td><td>D5637</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Boom</td><td>7047</td><td></td></tr><tr><td>Vectastain standard ABC-kit</td><td>Vector</td><td>PK6100</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>Urethane</td><td>Sigma</td><td>U2500</td><td></td></tr><tr><td>Isoflurane</td><td>Pharmachemie</td><td>45.112.110</td><td></td></tr><tr><td>Lidocaine</td><td>Sigma</td><td>L5647</td><td></td></tr><tr><td>Simplex rapid dental cement</td><td>Kemdent</td><td>ACR308/ACR924</td><td></td></tr><tr><td>Biocytin</td><td>Molekula</td><td>36219518</td><td></td></tr><tr><td>PFA</td><td>Merck Millipore</td><td>8187151000</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T4661</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Aldrich</td><td>81381</td><td></td></tr><tr><td>Analytical grade glycerol</td><td>Fluka</td><td>49767</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>31434</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>60130</td><td></td></tr><tr><td>CaCl</td><td>Sigma Aldrich</td><td>22,350-6</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka</td><td>63072</td><td></td></tr></tbody></table>

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

Universidad de Cartagena UDEC

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

科研

神经科学

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

大脑切片生物素化:一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

警报，头抑制大鼠子皮层的大脑结构中Juxtacellular监测和本地化单神经元

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

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

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

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

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

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

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

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

神经微电路急性脑片电生理和形态特征采用配对膜片钳记录

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

脊髓 Cocultures 丘脑-皮质轴突分支和突触形成的可视化研究

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

填充海马 ca2 间神经元的生物环素恢复与三维重建

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described ...

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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逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元