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>

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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 border="0" cellpadding="0" cellspacing="0" width="860">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Name of the Reagent</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Ketaset (Ketamine HCl)</td>
			<td style="width:179px;">Fort Dodge&#160;</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Anased (Xylazine solution)</td>
			<td style="width:179px;">Lloyd Laboratories</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Betadyne (Povidone-Iodine)</td>
			<td style="width:179px;">CVS Pharmacy</td>
			<td align="right" style="width:175px;">269281</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Loctite 495</td>
			<td style="width:179px;">Grainger Industrial Supply</td>
			<td style="width:175px;">4KL86</td>
			<td style="width:315px;">20-40 cp cyanoacrylate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Vetbond</td>
			<td style="width:179px;">3M</td>
			<td style="width:175px;">1469SB</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement powder</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675571</td>
			<td style="width:315px;">For the base of the recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Grip cement liquid</td>
			<td style="width:179px;">Dentsply Intl</td>
			<td align="right" style="width:175px;">675572</td>
			<td style="width:315px;">For the base of the recording chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Silicone Gel</td>
			<td style="width:179px;">Dow Corning</td>
			<td align="right" style="width:175px;">Mar-80</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:191px;">Jet denture repair acrylic powder</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:191px;">Ortho-Jet Fast curing orthodontic acrylic resin liquid</td>
			<td style="width:179px;">Lang Dental Manufacturing Co.</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">For securing the head restraint apparatus to the cranium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Chicago sky blue</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C8679</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Paraformaldehyde</td>
			<td style="width:179px;">Sigma</td>
			<td align="right" style="width:175px;">158127</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Phosphate-buffered saline</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">P3813</td>
			<td style="width:315px;">For perfusion and tissue fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:191px;">Cytochrome C</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">C2506</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Diaminobenzidine</td>
			<td style="width:179px;">Sigma</td>
			<td style="width:175px;">D5905</td>
			<td style="width:315px;">For cytochrome-oxidase staining (Figure 7)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Material Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:175px;">Catalogue Number</td>
			<td style="width:315px;">Comments&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Rat sock</td>
			<td style="width:179px;">Sew Elegant (San Diego, CA)</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 1, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">PVC tube 2 &#189;&#8221;</td>
			<td style="width:179px;">U.S. Plastic Co.</td>
			<td align="right" style="width:175px;">34108</td>
			<td style="width:315px;">Figure 4</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Subminiature D pins &#38; sockets</td>
			<td style="width:179px;">TE Connectivity</td>
			<td style="width:175px;">205089-1</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel music wire 0.010&#8221; diameter</td>
			<td style="width:179px;">Precision Brand Products, Inc.</td>
			<td align="right" style="width:175px;">21010</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic holding frame</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 900</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic ear bars</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 957</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Stereotaxic manipulator</td>
			<td style="width:179px;">Kopf Instruments</td>
			<td style="width:175px;">Model 960</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">&#189; mm drill burr</td>
			<td style="width:179px;">Henry Schein</td>
			<td style="width:175px;">100-3995</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quiet-Air dental drill&#160;</td>
			<td style="width:179px;">Midwest Dental</td>
			<td style="width:175px;">393-1600</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Stainless steel 0-80 1/8&#8221; screw</td>
			<td style="width:179px;">Fastener superstore</td>
			<td align="right" style="width:175px;">247438</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">0.2mL centrifuge tube</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:175px;">05-407-8A</td>
			<td style="width:315px;">Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Custom head-holding bar</td>
			<td style="width:179px;">UCSD SIO Machine Shop</td>
			<td style="width:175px;">N/A</td>
			<td style="width:315px;">Custom made, Figures 2, 3, 4</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:191px;">Custom head-holding plate</td>
			<td rowspan="2" style="width:179px;">UCSD SIO Machine Shop</td>
			<td rowspan="2" style="width:175px;">N/A</td>
			<td rowspan="2" style="width:315px;">Custom made, Figure 2, 3, 4</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Right angle post-clamp</td>
			<td style="width:179px;">Newport</td>
			<td style="width:175px;">MCA-1</td>
			<td style="width:315px;">Figure 3,4; standard opto-mechanical parts for the experimental jig (Figure 4) are also from Newport Corp.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">8-32 3/4&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">240181</td>
			<td style="width:315px;">For head-restraint, Figure 3</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">4-40 &#188;&#8221; screw</td>
			<td style="width:179px;">Fastener Superstore</td>
			<td align="right" style="width:175px;">239958</td>
			<td style="width:315px;">For head restraint, Figures 3, 4</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Quartz capillary tubing</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">QF-100-60-10</td>
			<td style="width:315px;">Figure 5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Carbon dioxide laser puller</td>
			<td style="width:179px;">Sutter instruments</td>
			<td style="width:175px;">P-2000</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Motorized micromanipulator</td>
			<td style="width:179px;">Sutter Instruments</td>
			<td style="width:175px;">MP-285</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">Multiclamp 700B</td>
			<td style="width:315px;">Alternate part: Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Microelectrode amplifier head stage</td>
			<td style="width:179px;">Molecular Devices</td>
			<td style="width:175px;">CV-7B</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:191px;">Isolated pulse stimulator</td>
			<td style="width:179px;">A-M Systems</td>
			<td style="width:175px;">Model 2100</td>
			<td style="width:315px;">Alternate part: HS-9Ax10 with Molecular Devices Axoclamp 900A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Audio monitor</td>
			<td style="width:179px;">Radio Shack</td>
			<td style="width:175px;">32-2040</td>
			<td style="width:315px;"></td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:191px;">Pipette holder</td>
			<td rowspan="2" style="width:179px;">Warner Instruments</td>
			<td rowspan="2" style="width:175px;">#MEW-F10T</td>
			<td style="width:315px;">Alternate parts: see Discussion</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:315px;">Figure 6A</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Electrode lead wire</td>
			<td style="width:179px;">Cooner wire</td>
			<td style="width:175px;">NEF34-1646</td>
			<td style="width:315px;">(optional), Figure 6D</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:191px;">Relay for amplifier head-stage</td>
			<td style="width:179px;">COTO Technology</td>
			<td style="width:175px;">#2342-05-000</td>
			<td style="width:315px;">(optional) Used with a custom-made printed circuit board (UCSD Physics Electronics Shop), Figure 6A-C</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:191px;">Digital video camera</td>
			<td style="width:179px;">Basler</td>
			<td style="width:175px;">A602fm</td>
			<td style="width:315px;">(optional) For behavioral monitoring, Figure 7</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

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

Single-cell Profiling of Developing and Mature Retinal Neurons

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and gene expression programs for extremely rare cell subsets has been a challenge using standard whole-tissue approaches. Gene expression profiling of individual cells allows for unprecedented access to cell types that comprise only a small percentage of the total tissue1-7. In addition, this technique can be used to examine the gene expression programs that are transiently expressed in small numbers of cells during dynamic developmental transitions8.
This issue of cellular diversity arises repeatedly in the central nervous system (CNS) where neuronal connections can occur between quite diverse cells9. The exact number of distinct cell types is not precisely known, but it has been estimated that there may be as many as 1000 different types in the cortex itself10. The function(s) of complex neural circuits may rely on some of the rare neuronal types and the genes they express. By identifying new markers and helping to molecularly classify different neurons, the single-cell approach is particularly useful in the analysis of cell types in the nervous system. It may also help to elucidate mechanisms of neural development by identifying differentially expressed genes and gene pathways during early stages of neuronal progenitor development.
As a simple, easily accessed tissue with considerable neuronal diversity, the vertebrate retina is an excellent model system for studying the processes of cellular development, neuronal differentiation and neuronal diversification. However, as in other parts of the CNS, this cellular diversity can present a problem for determining the genetic pathways that drive retinal progenitors to adopt a specific cell fate, especially given that rod photoreceptors make up the majority of the total retinal cell population11. Here we report a method for the identification of the transcripts expressed in single retinal cells (Figure 1). The single-cell profiling technique allows for the assessment of the amount of heterogeneity present within different cellular populations of the retina2,4,5,12. In addition, this method has revealed a host of new candidate genes that may play role(s) in the cell fate decision-making processes that occur in subsets of retinal progenitor cells8. With some simple adjustments to the protocol, this technique can be utilized for many different tissues and cell types.

A method for the isolation of single retinal cells and subsequent amplification of their cDNAs is described. Single-cell transcriptomics reveals the degree of cellular heterogeneity present in a tissue and uncovers new marker genes for rare cell populations. The accompanying protocol can be adjusted to suit many different cell types.

视网膜神经细胞发育和成熟的单细胞的剖析

Highly specialized, but exceedingly small populations of cells play important roles in many tissues. The identification of cell-type specific markers and ...

科研

神经科学

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

厚脑切片使用高尔基考克斯方法中成像神经元

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

Modeling Amyloid-&amp;#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

Transcranial Electrical Brain Stimulation in Alert Rodents

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in humans is transcranial direct current stimulation (tDCS). Less frequently, transcranial alternating current stimulation (tACS) or transcranial random noise stimulation (tRNS), a specific form of tACS using an electrical current applied randomly within a pre-defined frequency range, is used. The increase of noninvasive electrical brain stimulation research in humans, both for experimental and clinical purposes, has yielded an increased need for basic, mechanistic, safety studies in animals. This article describes a model for transcranial electrical brain stimulation (tES) through the intact skull targeting the motor system in alert rodents. The protocol provides step-by-step instructions for the surgical set-up of a permanent epicranial electrode socket combined with an implanted counter electrode on the chest. By placing a stimulation electrode into the epicranial socket, different electrical stimulation types, comparable to tDCS, tACS, and tRNS in humans, can be delivered. Moreover, the practical steps for tES in alert rodents are introduced. The applied current density, stimulation duration, and stimulation type may be chosen depending on the experimental needs. The caveats, advantages, and disadvantages of this set-up are discussed, as well as safety and tolerability aspects.

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact skull.

警惕啮齿动物的经颅电脑刺激

Transcranial electrical brain stimulation can modulate cortical excitability and plasticity in humans and rodents. The most common form of stimulation in ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

小鼠大脑中耳蜗的定位

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

A Bedside, Single Burr Hole Approach to Multimodality Monitoring in Severe Brain Injury

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic brain injury. While elevations in ICP are common, data regarding the measurement and treatment of these ICP elevations are conflicting. There is increasing recognition that changes in the balance between supply and demand of brain tissue are critically important and therefore the measurement of multiple modalities is required. Approaches are not standard, and therefore this article provides a description of a bedside, single burr hole approach to multimodality monitoring that allows the passage of probes designed to measure not only ICP but brain tissue oxygen, blood flow, and intracranial electroencephalography. Patient selection criteria, operative procedures, and practical considerations for securing probes during critical care are described. This method is readily performed, safe, secure, and flexible for the adoption of a variety of multimodality monitoring approaches aimed at detecting or preventing secondary brain injuries.

A method of recording multimodality monitoring signals in patients with severe brain injuries using a bedside, single burr hole technique is described.

一种用于重型颅脑损伤多模态监测的床边单毛刺孔方法

Intracranial pressure (ICP) monitoring is a cornerstone of the intensive care management of patients with severe acute brain injuries, including traumatic ...

Three-Dimensional Shape Modeling and Analysis of Brain Structures

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. We have developed a software package for accurate and robust shape modeling and group-wise analysis. Here, we introduce an pipeline for the shape analysis, from individual 3D shape modeling to quantitative group shape analysis. We also describe the pre-processing and segmentation steps using open software packages. This practical guide would help researchers save time and effort in 3D shape analysis on brain structures.

We introduce a semi-automatic protocol for shape analysis on brain structures, including image segmentation using open software, and further group-wise shape analysis using an automated modeling package. Here, we demonstrate each step of the 3D shape analysis protocol with hippocampal segmentation from brain MR images.

脑结构三维形状建模与分析

Statistical shape analysis of brain structures has been used to investigate the association between their structural changes and pathological processes. ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.