We are grateful to the Canadian Institutes of Health Research (grant MT-5877), the National Institutes of Health (grants NS058668 and NS066664), and the US-Israeli Binational Foundation (grant 2003222) for funding these studies.

实验方案进行了对女龙Evans大鼠（250 - 350克），根据联邦规定的动物护理和使用指南，并在加州大学圣地亚哥分校获得批准的机构动物护理和使用委员会。 1.在驯化老鼠身体约束注意:将大鼠在限制饮食。每日处理会话之后立即喂养的大鼠每天一次驯化大鼠的约束（以下描述）。提供足够的饲料，以保持动物在80％的初始重量。这一数额是大约6克每一天饲料250克重的雌龙埃文斯大鼠。 <ol><li>适应的老鼠被人类正在处理。轻轻抑制大鼠用手为约5秒，每30秒周期。做到这一点的15日连续2天分钟。每天监测大鼠应激训练期间的迹象。症状包括我挣扎ñ回应克制，发声，和齿抖动。 </li><li>在第3天，将大鼠在一身体约束布袜子（ 图1），用于在连续两天，每天15分钟。 </li><li>在第 5天，将老鼠的袜子内，并放置在袜子的刚性管上的试验夹具（ 图4）的内部。离开老鼠在管15日连续2天分钟。 </li><li>每次训练后立即喂老鼠。在上届会议（ 第 6天）结束给予不受限制地进入食物。选择植入只老鼠不表现出过度紧张的最后一个训练日的迹象。 注意:虽然选择大鼠植入是主观的，在我们手中大鼠的90％以上的被认为是响应于习惯和能够经历着床。 </li></ol> 2.植入的录音室和头部约束机制<ol><li>做一个重新ference丝通过焊接裸不锈钢丝以一个针连接器。剪断钢丝，使3-5毫米的遗体被发现的脚。高压灭菌的基准线，以及所有外科手术工具。 </li><li>管理氯胺酮/甲苯​​噻嗪麻醉。施用95毫克/千克氯胺酮与5mg / kg的甲苯噻嗪腹膜内混合。初始剂量持续1.5至2小时。补充麻醉以后每隔30-45分钟，视需要。润滑动物的眼睛的眼用软膏。 </li><li>检查脚趾撤退反射来确定麻醉平面，并维持麻醉是必要的。 </li><li>将老鼠耳酒吧立体保持框 （ 图3A）。剃的头部的毛发和消毒伤口部位与聚维酮碘（10％溶液）。照顾到插入耳棒适当地使它们不会损坏耳膜。耳棒用钝头为佳。 </li><li>做一个切口，用手术刀approxi三方共同沿从眼睛的rostro - 尾级的耳朵后面的头盖骨的中线。用剪刀剪下并除去皮肤的2-3毫米长条上的切口的任一侧。 </li><li>刮掉骨膜到颅骨的表面上暴露出来的横向脊。覆盖外露的头盖骨有薄薄的一层强力胶的。 </li><li>钻一小孔，用直径略小于0-80螺丝，采用了半厘米直径的钻孔毛刺（见材料部分）。钻孔立即后侧到前囟门缝合满足横向脊，和在一30-45°角插入颅骨，以便螺杆可以在与顶部比底部更外侧。 </li><li>拧在0-80平底机螺丝孔（大约3个圈），在30-45°角。要小心，不要拧得太深，以免损坏底层的脑组织。 </li><li>重复此过程所示配置6个额外的螺丝<strong&gt;（ 图3B）。应用强力胶的所有螺丝底座。 </li><li>将注射器针头在stereotax操纵。从囟缝合测量，并在与邻近针头的头盖骨的凹痕，但外面的所需开颅位置的，作为一个基准标记。 注:在本演示中，该商标的立体坐标3毫米后1毫米横向到前囟门缝合。 </li><li>标志着一个永久性标记的凹痕。注意商标的立体坐标，因为它会成为一个立体的参考点 （ 图3B）。 </li><li>使集中于所希望的坐标（3毫米后部和3mm的横向在这个例子中，以前囟）开颅手术。离开硬膜保持完整。覆盖改进的人工脑脊髓液的开颅（125 mM氯化钠，10mM葡萄糖，10mM的HEPES，3.1毫摩尔氯化钙 ，1.3毫的MgCl 2，pH7.4）中13（ 图3C）。 </li&gt; <li>切0.2毫升离心管中，以4 - 5毫米长度，并切断帽。将管的头盖骨和居中在开颅手术。 </li><li>申请围绕管的底部牙科用粘固剂，以密封所述管到头盖骨的底部。要小心，不要漏水泥进入开颅暴露（ 图3D）。 </li><li>另一个钻小孔（约半毫米直径）对侧颅板，并小心将参考线。通过焊接的销，与记录放大器向所述导线的端部对接构造的基准线（见设备部分）。不要移动参考线，一旦它在大脑中，因为这可能会造成的损害。 </li><li>应用强力胶的丝被插入的孔。这将封针和线的地方暂时的。 </li><li>混合硅凝胶试剂盒的两部分（参见材料节）在大约相等的部分。等待两分钟，混合和FIL升离心管大约⅓充满凝胶。 </li><li>附加直角钳后（见材料部分）的头枕杆。以45°倾斜角连接头板（ 图2A）的固定条（ 图2B），使得板的底部是朝动物的鼻子。是有帮助的使用倾斜计来设定倾斜角以匹配实验夹具（ 图4）的。 </li><li>附加的固定条的stereotax操纵臂，使杆平行于耳棒。降低杆和板，使该板是在lambda缝合和前向尾最螺杆后部。 </li><li>抓住前头螺栓（8-32螺柱或螺钉，参见材料节）在约45°角与帮助手臂和夹紧，从而使螺钉的头部朝下，朝向动物的尾巴。降低螺丝，这样倒是蚂蚁erior 0-80螺丝 （ 图3F，G）。 </li><li>固定到位的螺钉和钢板与牙科丙烯酸。申请围绕骨螺钉头上的牙科丙烯酸，基准销，和周围的离心管的两侧。等待大约10分钟的牙科丙烯酸干 （ 图3H）。 </li><li>敷一层牙科水泥周围牙科丙烯酸的边缘，固井皮肤给植入物。等待水泥干。 </li><li>戳几个洞，在离心管的盖子，并放置在管帽。 </li><li>取下援助之手钳。然后小心地取出从stereotax头控股栏，然后在酒吧（ 图​​3I）去除头板。 </li><li>从stereotax取出的动物和管理术后护理和监测符合所有适用的法规，规章和法律（ 如丁丙诺啡0.02毫克/公斤，每8-12小时最少24小时）。如果炎症围绕所述植入物的边缘的开发，应用一薄层抗生素软膏到患部每日直至解决。 </li></ol>神经元的单位3. Juxtacellular监控<ol><li>拉动一个二氧化碳激光微量拉马石英毛细管（见设备部分）具有小于1微米（ 图5A）尖端直径。 注:在加热参数将根据特定仪器而变化，并且，依赖于所关心的大脑结构所需的颈部直径会有所不同。对于录音大鼠丘脑，有微量的脖子，从5-7毫米。 </li><li>固定到位的吸管微分干涉对比（DIC）显微镜配有长工作距离的目标之下。用橡皮泥举行吸管的地方在显微镜的阶段。 </li><li>缓慢移动的玻璃块（0.5 - 1厘米厚;用于MAK一块玻璃荷兰国际集团超薄切片机刀是方便）进入视用吸管尖端的领域。使用阶段显微，轻轻触摸枪头玻璃，使其打破。直到枪头外径为1-3微米之间根据需要重复 （ 图5B，C）。确保这些微量介于约5-15MΩ阻抗。 </li><li>制备的细胞外液（135 mM氯化钠，5.4 mM的氯化钾，1.8mM的氯化钙 ，1mM的MgCl 2的，5mM的HEPES，pH值7.2，用NaOH）8，并添加2％（重量/体积）芝加哥天蓝。使用注射器与地下30针或更小的，填充的溶液的吸液管的后端。可替代地，对于单细胞juxtacellular标记添加2％（重量/体积）Neurobiotin或生物素化葡聚糖胺（BDA-3000或BDA-10000），以代替芝加哥天蓝的盐溶液。 </li><li>放置在刚性管内的布袜子内的老鼠在适当的实验夹具 （ <strong&gt;的图4）.Wait手术后至少72小时的放置动物的头部约束夹具之前。收紧束带围绕上部胸骨，而不是在脖子上，以避免阻塞气道。如果呼吸似乎吃力或阻塞，松开拉绳或将其向后。开展这一程序，而老鼠是警报。 </li><li>连接头压紧板对大鼠上的夹具的相应部件。要做到这一点，第一脚头克制板的地方，然后用4-40螺丝将其固定。一定要等到动物是平静，以避免施加过大的扭矩头部。下一步，放置8-32螺母被植入大鼠头部约束螺栓。再拧在带螺纹的不锈钢棒的头部的螺栓。贴上所述杆的实验夹具以这样一种方式，它可以在适当位置紧固（ 图4）。 注:保护与头螺栓的动物以及头部约束板分imizes装置的弯曲，提高了记录的稳定性。在大多数情况下，记录可以在第一天开始，动物是头抑制。但是，如果过分动物坐立不安或大谈，在开始任何录音之前提供一天头部约束习惯的培训。在这种情况下，留在夹具的动物为15分钟，然后将它放回其笼子。重复此步骤，次日并继续协议。 </li><li>打开录音室，取出硅胶。用干净细镊子，如果组织已重新种植在开颅开颅手术。 </li><li>附上吸管电动显微，并插上探头前置放大器。 注意:在本示范，在探头小型继电器电路用于放大器引线和外部电流源以高顺应性（ 图6A-C）之间切换。请注意，某些放大器内置适当的依从性高源（见讨论和材料的部分）。 </li><li>使用电动显微到移液管的尖端移动到立体定位识别标记的录音室中。注意此位置的坐标。要小心，不要打破颅骨的吸管尖。 </li><li>移动的吸移管向期望的记录位置，在前后和内 - 外轴。然后前进移液器腹侧通过硬脑膜，直到它是约500微米背侧到预定记录位置。 </li><li>一边听的枪头和参考线之间的记录放大电压的音频监视器扣球事件慢慢推进吸管。一旦扣球事件被确定，继续往前移液管0-100微米，直到正向电压偏差大于约500μV观察。 注:电极电阻将约为1.5-10 WH因素增加恩发生这种情况。 </li><li>开始一次一个单元已被分离为根据步骤3.11，连同所有感兴趣的其它行为和生理措施记录。在本示范，自生触须动作都与高速摄像（参见材料节）进行监测。 </li><li>标记记录站点，切换电极引线连接到电流源。有几种方法可以做到这一点，使用一个继电器电路（ 图6A - C）中，一个内置的电流源上的放大器，或手动（ 图6D，参见步骤3.8和讨论 ）。传递-4μA与2秒的脉冲，在50％的占空比为4分钟，通过吸液管以离子电渗注入芝加哥天蓝。 </li><li>杀死并根据标准的做法几经标签的程序灌注动物。部分根据需要大脑和染液以识别记录的解剖位置坐即染液组织细胞色素氧化酶的活性按14。另外，确定使用荧光显微镜的芝加哥天空蓝存款。 注:为不同的标记单元之间的准确区分是很重要的，所有的标签都是用在同一天同一个吸管，无需松开标签之间的吸管。在这种情况下，记录的站点可以通过它们的相对位置在事后组织学以及每个站点的注意操纵器的坐标来区分（参见步骤3.9）。对于明确标识，从彼此，没有每个大脑区域超过3-5标签标注的标签，至少200微米分开。 </li></ol>

<ol>
	<li>Fee, M. S., Leonardo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniature+motorized+microdrive+and+commutator+system+for+chronic+neural+recording+in+small+animals.">Miniature motorized microdrive and commutator system for chronic neural recording in small animals.</a> Journal of Neuroscience Methods. 112 (2), 83-94 (2001).</li><li>Ventakachalam, S., Fee, M. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-miniature+headstage+with+6-channel+drive+and+vacuum-assisted+micro-wire+implantation+for+chronic+recording+from+neocortex.">Ultra-miniature headstage with 6-channel drive and vacuum-assisted micro-wire implantation for chronic recording from neocortex.</a> Journal of Neuroscience Methods. 90 (1), 37-46 (1999).</li><li>Szuts, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wireless+multi-channel+neural+amplifier+for+freely+moving+animals.">A wireless multi-channel neural amplifier for freely moving animals.</a> Nature Neuroscience. 14 (2), 263-269 (2011).</li><li>Roy, S., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wireless+multi-channel+single+unit+recording+in+freely+moving+and+vocalizing+primates.">Wireless multi-channel single unit recording in freely moving and vocalizing primates.</a> Journal of neuroscience. 203 (1), 28-40 (2012).</li><li>Hill, D. N., Mehta, S. B., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+metrics+to+accompany+spike+sorting+of+extracellular+signals.">Quality metrics to accompany spike sorting of extracellular signals.</a> Journal of Neuroscience. 31 (24), 8699-8705 (2011).</li><li>Pinault, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+single-cell+staining+procedure+performed+in+vivo+under+electrophysiological+control:+morpho-functional+features+of+juxtacellularly+labeled+thalamic+cells+and+other+central+neurons+with+biocytin+or+Neurobiotin.">A novel single-cell staining procedure performed in vivo under electrophysiological control: morpho-functional features of juxtacellularly labeled thalamic cells and other central neurons with biocytin or Neurobiotin.</a> Journal of neuroscience. 65 (2), 113-136 (1996).</li><li>Person, A. L., Perkel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pallidal+neuron+activity+increases+during+sensory+relay+through+thalamus+in+a+songbird+circuit+essential+for+learning.">Pallidal neuron activity increases during sensory relay through thalamus in a songbird circuit essential for learning.</a> The Journal of neuroscience. 27 (32), 8687-8698 (2007).</li><li>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=Spiking+in+primary+somatosensory+cortex+during+natural+whisking+in+awake+head-restrained+rats+is+cell-type+specific.">Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific.</a> Proceedings of the National Academy of Sciences USA. 106 (38), 16446-16450 (2009).</li><li>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), 10481061 (2010).</li><li>Hellon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marking+of+electrode+tip+positions+in+nervous+tissue.">The marking of electrode tip positions in nervous tissue.</a> The Journal of physiology. 214, 12P (1971).</li><li>Furuta, T., Desch&#234;nes, M., Kaneko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+distribution+of+thalamocortical+boutons+in+barrels.">Anisotropic distribution of thalamocortical boutons in barrels.</a> The Journal of Neuroscience. 31 (17), 6432-6439 (2011).</li><li>Paxinos, G., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Brain in Stereotaxic Coordinates. , (1986).</li><li>Kleinfeld, D., Delaney, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distributed+representation+of+vibrissa+movement+in+the+upper+layers+of+somatosensory+cortex+revealed+with+voltage+sensitive+dyes.">Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage sensitive dyes.</a> Journal of Comparative Neurology. 375 (1), 89-108 (1996).</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 research. 171 (1), 11-28 (1979).</li><li>Moore, J. D., Desch&#234;nes, M., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-generated+vibrissa+motion+and+touch+are+differentially+represented+throughout+ventral+posterior+medial+thalamus+in+awake,+head-fixed+rats.">Self-generated vibrissa motion and touch are differentially represented throughout ventral posterior medial thalamus in awake, head-fixed rats.</a> Society for Neuroscience Annual Meeting. 41 496.08/TT424 Society for Neuroscience. 41, (2011).</li><li>Khatri, V., Bermejo, R., Brumberg, J. C., Zeigler, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whisking+in+air:+Encoding+of+kinematics+by+VPM+neurons+in+awake+rats.">Whisking in air: Encoding of kinematics by VPM neurons in awake rats.</a> Somatosensory and Motor Research. 27 (2), 344-356 (2010).</li><li>Hill, D. N., Curtis, J. C., Moore, J. D., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+motor+cortex+reports+efferent+control+of+vibrissa+position+on+multiple+time+scales.">Primary motor cortex reports efferent control of vibrissa position on multiple time scales.</a> Neuron. 72 (2), 344-356 (2011).</li><li>Moore, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchy+of+orofacial+rhythms+revealed+through+whisking+and+breathing.">Hierarchy of orofacial rhythms revealed through whisking and breathing.</a> Nature. 497, 205-210 (2013).</li><li>Duque, A., Zaborszky, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomical Tract-Tracing 3. , 197-236 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroactive substances: Neuropharmacology by microiontophoresis. , .</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dyes and Tracers: Sitemarking and tracktracing by microiontophoresis. , .</li><li>Urbain, N., Desch&#234;nes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroactive+substances:+Neuropharmacology+by+microiontophoresis.+(Kation+Scientific),+Dyes+and+Tracers:+Sitemarking+and+tracktracing+by+microiontophoresis.+(Kation).">Neuroactive substances: Neuropharmacology by microiontophoresis. (Kation Scientific), Dyes and Tracers: Sitemarking and tracktracing by microiontophoresis. (Kation).</a> Journal of Neuroscience. 27 (45), 12407-12412 (2007).</li></ol>

作者宣称，他们有没有竞争的财务权益。

试验夹具的建设 用于构建实验夹具（ 图4）的机械部件的描述从协议中省略，因为它可以以各种方式来构造。在此示范标准光学机械部件和支撑夹具用于安装头枕杆和身体约束管（参见材料节）。类似光学机械零件可以被用来在电极保持器安装到机动显微。重要的是在夹具的头部约束杆被安装在相同的螺距角所用外科手术期间...

监测神经元活动中积极从事一个行为的任务的警报动物对理解神经系统的功能和组织的关键。从单个神经元单位电活动的细胞外记录一直是神经系统的主食工具，仍然是广泛使用在目前。各种电极类型和配置都可以根据具体实验的科学和技术要求。长期植入微型硬盘或电极阵列通常在自由活动的动物，包括鸟类，啮齿类动物，非人灵长类动物1-4使用。可替代地，通过外部显微急性穿透与金属或玻璃微电极通常用于从麻醉或头部限制动物来记录。玻璃微电极具有它们可以被用在juxtacellular或"细胞附着"的优势构明确地隔离单个神经元无事后秒杀分拣5的并发症的活性。这些电极从标题解剖的细胞或位置进一步允许记录，因为它们可以被用于注入的染料或神经解剖学示踪剂小存款，或甚至填充单个记录单元。这种配置已经在老鼠和鸟类6-10得到成功应用。目前所描述的技术，专注于juxtacellular监测和细胞外染料存款警报，头克制老鼠。注意juxtacellular罢了，这些染料存款不提供有关细胞形态和轴突的突起11的信息，不像单个细胞，但它们能精确解剖定位到约50微米，关键的是，有显著产量较高的警觉动物。对于单细胞juxtacellular填充仍然是作为用于解剖标签替代战略的信息。 简言之，在协议包括三个主要阶段。在第一阶段，将大鼠驯化身体约束在一个布袜子（ 图1）历时6天。在第二阶段中，头枕装置（ 图2）和记录室被手术植入，使得老鼠可以在多个后续的记录会话（ 图3）被保持在立体平面上;这个程序可以使实验者基于标准的参考坐标12为目标的大脑的特定子皮层区域的电生理研究。第三阶段涉及将大鼠在适当的夹具用于进行行为和电生理实验（ 图4），从一个石英毛细管构成电极（ 图5），使得juxtacellular神经元的录音是明确分离单个单元6-9，和标志着解剖LOCATI在记录现场与芝加哥天蓝染料（ 图6和7）组成。在录音同声行为进行监督;然而，该行为的技术细节将取决于每个实验的科学目标，因而超出了单个协议的范围。的实验程序，它可以被重复多天完成后，将动物安乐死。大脑是根据使用或者明场或荧光显微镜标准神经解剖技术开采和加工。

<table><tbody><tr><td>Ketaset (Ketamine HCl)</td><td>Fort Dodge</td><td>N/A</td><td></td></tr><tr><td>Anased (Xylazine solution)</td><td>Lloyd Laboratories</td><td>N/A</td><td></td></tr><tr><td>Betadyne (Povidone-Iodine)</td><td>CVS Pharmacy</td><td>269281</td><td></td></tr><tr><td>Loctite 495</td><td>Grainger Industrial Supply</td><td>4KL86</td><td>20 - 40 cp cyanoacrylate</td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>Grip cement powder</td><td>Dentsply Intl</td><td>675571</td><td>For the base of the recording chamber</td></tr><tr><td>Grip cement liquid</td><td>Dentsply Intl</td><td>675572</td><td>For the base of the recording chamber</td></tr><tr><td>Silicone gel</td><td>Dow Corning</td><td>3-4680</td><td></td></tr><tr><td>Jet denture repair acrylic powder</td><td>Lang Dental Manufacturing Co.</td><td>N/A</td><td>For securing the head restraint apparatus to the cranium</td></tr><tr><td>Ortho-Jet Fast curing orthodontic acrylic resin liquid</td><td>Lang Dental Manufacturing Co.</td><td>N/A</td><td>For securing the head restraint apparatus to the cranium</td></tr><tr><td>Chicago sky blue</td><td>Sigma</td><td>C8679</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>For perfusion and tissue fixation</td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma</td><td>P3813</td><td>For perfusion and tissue fixation</td></tr><tr><td>Cytochrome C</td><td>Sigma</td><td>C2506</td><td>For cytochrome-oxidase staining, &lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr><tr><td>Diaminobenzidine</td><td>Sigma</td><td>D5905</td><td>For cytochrome-oxidase staining, &lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr><tr><td>Rat sock</td><td>Sew Elegant (San Diego, CA)</td><td>N/A</td><td>Custom made, &lt;strong&gt;Figures 1, 4&lt;/strong&gt;</td></tr><tr><td>PVC tube 2 &amp;frac12;&amp;rdquo;</td><td>U.S. Plastic Co.</td><td>34108</td><td>&lt;strong&gt;Figure 4&lt;/strong&gt;</td></tr><tr><td>Subminiature D pins &amp;amp; sockets</td><td>TE Connectivity</td><td>205089-1</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>Stainless steel music wire 0.010&amp;rdquo; diameter</td><td>Precision Brand Products, Inc.</td><td>21010</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>Stereotaxic holding frame</td><td>Kopf Instruments</td><td>Model 900</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>Stereotaxic ear bars</td><td>Kopf Instruments</td><td>Model 957</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>Stereotaxic manipulator</td><td>Kopf Instruments</td><td>Model 960</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>&amp;frac12; mm drill burr</td><td>Henry Schein</td><td>100-3995</td><td></td></tr><tr><td>Quiet-Air dental drill</td><td>Midwest Dental</td><td>393-1600</td><td></td></tr><tr><td>Stainless steel 0-80 &amp;frac18;&amp;rdquo; screw</td><td>Fastener superstore</td><td>247438</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>0.2 ml centrifuge tube</td><td>Fisher Scientific</td><td>05-407-8A</td><td>&lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>Custom head-holding bar</td><td>UCSD SIO Machine Shop</td><td>N/A</td><td>Custom made, &lt;strong&gt;Figures 2 - 4&lt;/strong&gt;</td></tr><tr><td>Custom head-holding plate</td><td>UCSD SIO Machine Shop</td><td>N/A</td><td>Custom made, &lt;strong&gt;Figures 2 - 4&lt;/strong&gt;</td></tr><tr><td>Right angle post-clamp</td><td>Newport</td><td>MCA-1</td><td>&lt;strong&gt;Figures 3, 4&lt;/strong&gt;; standard opto-mechanical parts for the experimental jig (&lt;strong&gt;Figure 4&lt;/strong&gt;) are also from Newport Corp.</td></tr><tr><td>8-32 &amp;frac34;&amp;rdquo; screw</td><td>Fastener Superstore</td><td>240181</td><td>For head-restraint, &lt;strong&gt;Figure 3&lt;/strong&gt;</td></tr><tr><td>4-40 &amp;frac14;&amp;rdquo; screw</td><td>Fastener Superstore</td><td>239958</td><td>For head restraint, &lt;strong&gt;Figures 3, 4&lt;/strong&gt;</td></tr><tr><td>Quartz capillary tubing</td><td>Sutter Instruments</td><td>QF-100-60-10</td><td>&lt;strong&gt;Figure 5&lt;/strong&gt;</td></tr><tr><td>Carbon dioxide laser puller</td><td>Sutter instruments</td><td>P-2000</td><td></td></tr><tr><td>Motorized micromanipulator</td><td>Sutter Instruments</td><td>MP-285</td><td></td></tr><tr><td>Microelectrode amplifier</td><td>Molecular Devices</td><td>Multiclamp 700B</td><td>Alternate part: Molecular Devices Axoclamp 900A</td></tr><tr><td>Microelectrode amplifier head stage</td><td>Molecular Devices</td><td>CV-7B</td><td>Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A</td></tr><tr><td>Isolated pulse stimulator</td><td>A-M Systems</td><td>Model 2100</td><td>Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A</td></tr><tr><td>Audio monitor</td><td>Radio Shack</td><td>32-2040</td><td></td></tr><tr><td>Pipette holder</td><td>Warner Instruments</td><td>#MEW-F10T</td><td>Alternate parts: see &lt;strong&gt;Discussion&lt;/strong&gt;, &lt;strong&gt;Figure 6A&lt;/strong&gt;</td></tr><tr><td>Electrode lead wire</td><td>Cooner wire</td><td>NEF34-1646</td><td>(optional), &lt;strong&gt;Figure 6D&lt;/strong&gt;</td></tr><tr><td>Relay for amplifier head-stage</td><td>COTO Technology</td><td>#2342-05-000</td><td>(optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), &lt;strong&gt;Figure 6A-C&lt;/strong&gt;</td></tr><tr><td>Digital video camera</td><td>Basler</td><td>A602fm</td><td>(optional) For behavioral monitoring, &lt;strong&gt;Figure 7&lt;/strong&gt;</td></tr><tr><td>Puralube vet ointment</td><td>Amazon.com, Inc</td><td>NC0138063</td><td></td></tr></tbody></table>

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.

<html>神经元单元中腹后内侧（VPM）丘脑编码触须移动的相位时自生搅打15,16。 图7A示出一个VPM丘脑单元的样品尖峰活性大鼠正在积极地搅拌。 图7B示出穗的直方图次对齐触须运动17的瞬时相位。有在搅打的缩回阶段更尖峰。在记录之后，本机的位置是通过芝加哥天蓝染料离子导入标记， 如图7C所示 。该组织counterstained细胞色素氧化酶的活性，?...

Watch this Scientific Journal Video about Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats at JoVE.com

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

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 ...

juxtacellular-monitoring-localization-single-neurons-within-sub

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.3K 次观看.  University of California, San Diego. 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.

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

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal&#8217;s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

This method creates a tangible, familiar environment for the mouse to navigate and explore during microscopic imaging or single-cell electrophysiological recordings, which require firm fixation of the animal&#8217;s head.

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a ...

科研

行为学

Recording Large-scale Neuronal Ensembles with Silicon Probes in the Anesthetized Rat

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of behaviors from the spiking activity of its neurons. One of the most effective methods to monitor spiking activity from a large number of neurons in multiple local neuronal circuits simultaneously is by using silicon electrode arrays1-3.
Action potentials produce large transmembrane voltage changes in the vicinity of cell somata. These output signals can be measured by placing a conductor in close proximity of a neuron. If there are many active (spiking) neurons in the vicinity of the tip, the electrode records combined signal from all of them, where contribution of a single neuron is weighted by its 'electrical distance'. Silicon probes are ideal recording electrodes to monitor multiple neurons because of a large number of recording sites (+64) and a small volume. Furthermore, multiple sites can be arranged over a distance of millimeters, thus allowing for the simultaneous recordings of neuronal activity in the various cortical layers or in multiple cortical columns (Fig. 1). Importantly, the geometrically precise distribution of the recording sites also allows for the determination of the spatial relationship of the isolated single neurons4. Here, we describe an acute, large-scale neuronal recording from the left and right forelimb somatosensory cortex simultaneously in an anesthetized rat with silicon probes (Fig. 2).

Extracellular recordings of neuronal activity using silicon probes in the anesthetized rat will be described. This technique allows information to be obtained across multiple brain areas from more than 100 neurons simultaneously. It provides information with single cell resolution about neuronal ensembles dynamics in multiple local circuits.

在麻醉大鼠的硅探头录制的大型神经元合唱

Large scale electrophysiological recordings from neuronal ensembles offer the opportunity to investigate how the brain orchestrates the wide variety of ...

神经科学

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical neurons with two-photon microscopy has provided unique insights into cortical structure, function and plasticity. However, these studies are limited to head fixed animals, greatly reducing the behavioral complexity available for study. In this paper, we describe a procedure for performing chronic fluorescence microscopy with cellular-resolution across multiple cortical layers in freely behaving mice. We used an integrated miniaturized fluorescence microscope paired with an implanted prism probe to simultaneously visualize and record the calcium dynamics of hundreds of neurons across multiple layers of the somatosensory cortex as the mouse engaged in a novel object exploration task, over several days. This technique can be adapted to other brain regions in different animal species for other behavioral paradigms.

Multi-layer Cortical Ca2+ Imaging in Freely Moving Mice with Prism Probes and Miniaturized Fluorescence Microscopy

Here, we present a procedure for performing large-scale Ca2+ imaging with cellular-resolution across multiple cortical layers in freely moving mice. Hundreds of active cells can be observed simultaneously using a miniature, head-mounted microscope coupled with an implanted prism probe.

多层皮质Ca<sup&gt; 2+</sup&gt;使用棱镜探针和小型荧光显微镜成像自由移动的小鼠

In vivo circuit and cellular level functional imaging is a critical tool for understanding the brain in action. High resolution imaging of mouse cortical ...

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

在啮齿类动物的存活立体定向手术

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

生物学

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

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

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 ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

急性<em&gt;体内</em&gt;麻醉大鼠超导途径局部场电位和多单位活动的电生理记录

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

使用微棱镜对头部固定和自由移动的动物进行多层成像

Long-Term Imaging of Identified Neural Populations using Microprisms in Freely Moving and Head-Fixed Animals

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for studying the structure, function, and plasticity of living brain tissues. In comparison to conventional electrophysiology, fluorescence microscopy can capture the neural activity as well as the morphology of the cells, enabling long-term recordings of the identified neuron populations at single-cell or subcellular resolution. However, high-resolution imaging typically requires a stable, head-fixed setup that restricts the movement of the animal, and the preparation of a flat surface of transparent glass allows visualization of neurons at one or more horizontal planes but is limited in studying the vertical processes running across different depths. Here, we describe a procedure to combine a head plate fixation and a microprism that gives multilayer and multimodal imaging. This surgical preparation not only gives access to the entire column of the mouse visual cortex but allows two-photon imaging in a head-fixed position and one-photon imaging in a freely moving paradigm. Using this approach, one can sample identified cell populations across different cortical layers, register their responses under head-fixed and freely moving states, and track the long-term changes over months. Thus, this method provides a comprehensive assay of the microcircuits, enabling direct comparison of neural activities evoked by well-controlled stimuli and under a natural behavioral paradigm.

作者聚焦:清醒和自由移动状态下神经活动的比较成像

When integrated with a head-plate and an optical design compatible with both single- and two-photon microscopes, the microprism lens presents a significant advantage in measuring neural responses in a vertical column under diverse conditions, including well-controlled experiments in head-fixed states or natural behavioral tasks in freely moving animals.

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

With the advancement of multi-photon microscopy and molecular technologies, fluorescence imaging is rapidly growing to become a powerful approach for ...

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

摘要

探索更多视频

此视频中的章节

平地板的空气解禁平台:一种新方法与显微镜或电对清醒结合行为自由移动鼠害

在麻醉大鼠的硅探头录制的大型神经元合唱

多层皮质Ca

在啮齿类动物的存活立体定向手术

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

急性

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像

在自由移动和头部固定的动物中使用微棱镜对已识别的神经种群进行长期成像