居里夫人国际传出奖学金（欧盟的FP/2007-2013的赠款协定，＃221834和254780），JD麦克唐纳基金会NSF资助SBE 0542013，国家格兰特NS034994卫生研究院，国家格兰特MH5467心理健康研究所和霍华德休斯医学研究所（Janelia农场研究校园补助金）。

1。建设的Microdrive 所有驱动器是由相同的基本元素：运动的一部分，它带有电极和一个固定的部分，这是固定的头骨。一个理想的微硬盘允许平稳，但长期在多个小步骤的电极足够的旅行，是足够坚固，以​​防止意外移动电极，容易操作，而不会干扰动物的行为，具有体积小，重量轻的实验者。由于这些竞争要求的结果，不同的驱动器套件不同的应用程序。 只有4个部分，需要建立我们的基本驱动器：黄铜平头螺钉，匹配的螺母，塑料桥，从单排排针和两个自定义切黄铜板的准备。 <ol><li>打破了头3针片</li><li>轻轻拉出中间的脚。 </li><li>放大通过它与钻头尺寸＃55钻洞。 </li><li>剪下一个TH阅读使用00-90自来水。 </li><li>切出两块铜板。 </li><li>文件边缘与Dremmel板。 </li><li>钻一个孔在中间的两件用钻头尺寸＃65。 </li><li>使铜板接触的针脚装配传动件。要做到这一点，插入黄铜螺丝，先后，铜板，脚头的螺纹孔，第二个铜板，螺母。轻轻拧紧螺丝，使大会成为稳定。 </li><li>焊接引脚结束铜板。 </li><li>文件中的螺丝凸出。 </li><li>焊接螺母螺丝。要小心，不要焊接铜板螺母。 </li><li>测试驱动器的运动：把螺丝顺时针，以提高塑料的桥梁。 </li></ol> 2。准备的硅探针固定探头驱动器之前，添加额外的绝缘探头粘接面积，以防止cerebrospinal脑脊液（CSF）或产生短路，湿度： <ol><li>权衡和10:1的比例混合Sylgard弹性元件。 </li><li>使用削尖的棉花撒施，适用于高端探头的Sylgard。 </li><li>在60°C时为2小时预热烤箱，让它干。 </li></ol>为了确保录音的网站是没有任何碎片，探针需要清洗： <ol start="4"><li>准备一个Contrad洗涤剂稀释4％。 </li><li>让探头洗涤剂浸泡在63°C至少2小时。 </li><li>探头反复浸泡在蒸馏水冲洗洗涤剂。 </li></ol>固定探头驱动器之前，每个录制现场的阻抗应检查： <ol start="7"><li> 0.9％的盐水中，浸探头，连接阻抗米。如果有太多的记录点阻抗不正确，请重复步骤2.4-2.6，或考虑使用不同的探头。在这里，我们使用阻抗调理模块，冯检基Haer有限公司（金控）相结合，用自制的通道选择。另外，，由NeuroNexus，公司或NanoZ的公司，由Neuralynx，niPOD允许阻抗所有的探头通道同时监测。 </li></ol> 3。贴上探头的Microdrive <ol><li>用刀片切成多个沟槽的桥梁，建立一个坚固的表面。 </li><li>放在附近探测到驱动桥。这个过程最好是在手术显微镜下，驱动器与钳和调整探头，通过显微操作，使柄与传动螺杆完全平行。这将确保在进步，进入脑组织的探头柄举动没有“切割通过它。在这个阶段，考虑到目标结构的深度，从表面探针驱动器的基础相对确切的深度应确定头骨。 </li><li>然后将探头固定桥握水泥。 </li><li>可选：在大脑中的可视化探测轨道，DII的解决方案（1-2％的乙醇稀释），可应用在这个阶段，到探头的背面。 </li></ol> 4。准备头骨手术前，参考和接地电极，在头法拉第笼的部分准备： <ol><li>削减两个铜线2“长的小段，和焊料各约1毫米的绝缘铜线。 </li><li>用针，刮头1 00-90，1/8“不锈钢螺丝和焊接一块​​铜线焊接等不锈钢接地螺钉电极需要一个适当的流量（例如，N-3目的从La有限公司通量）和烙铁头温度高。小心防止任何焊料流入螺杆的凹槽，这将作为接地电极使用。重复另一个螺钉和铜线准备参比电极。 </li><li>削减梯形铜网片。这些作品将被组装保护探头。 </li></ol>手术器械和准备许多小动物手术中使用的相同。整个手术做深异氟醚麻醉下，用无菌条件下，根据国立卫生研究院批准的指引。请注意，仅用于演示目的是在这部影片中（模拟）手术。对于适当的知名度和拍摄的目的，几个准备步骤，手术注意事项和术后的程序不显示/可见或讨论。 手术前，所有的部件和用品应消毒，适当程序后（生存鼠类手术的指引; http://oacu.od.nih.gov/ARAC/surguide.pdf）。在手术过程中，准备对头骨的无菌区和隔离无菌单。在结束手术，广谱抗生素是pplied本地和长效止痛药，肌肉注射（例如，丁丙诺啡，Buprenex] 0.05毫克/千克）。此外，止痛药（如布洛芬）提供饮用水，约60 mg/kg/24小时5天。对于适当的手术和麻醉的程序，征询适当的来源12。 <ol start="4"><li>安装在立体定位仪动物，剃须和清洁头皮13。 </li><li>削减沿中线的皮肤，拨开头皮。取下骨膜，清洁和干燥的头骨。 </li><li>测量前囟门和lambda之间的位置和距离，并确定相应采用立体定向图谱14探针植入网站的x和y坐标。刮用手术刀在十字架上的头骨，标志着网站。 </li><li>在上面钻颅骨成骨中途使用一个圆形头钻头（大小的四分之一）和驱动器支持螺钉（不锈钢，000-120，1/16“），对不同的骨板，Nd对头骨的一侧。螺丝将提供安全债券的头饰头骨的锚。 </li><li>上述小脑钻洞并插入地面和步骤4.2准备的参考电极。记录局部场电位（LFP），参考网站的选择是至关重要的。选择这个网站是因为小脑LFP的所有皮层区域和肌肉文物中最小的，这正中位置的最小。 </li><li>应用牙本质激活（Metabond包），使用小刷子在整个表面的头骨。 0.9％的生理盐水冲洗。 </li><li>适用于牙科水泥（Metabond套件;按照制造商的指示进行搅拌）的头骨，仔细覆盖锚螺钉和接地参考电极，但离开探针植入网站的明确。 </li><li>获得四铜网皮瓣（步骤4.3准备）的头骨。对于这一点，巩固他们每个人的狭隘基地前，左，右，后头骨两侧。日é铜绝不应与骨直接接触，但总是由水泥层分离。 </li></ol> 5。准备在大脑表面<ol><li>用圆头钻头，钻植入部位周围的多个阶段，而经常灌溉用生理盐水骨。 </li><li>小心地取下骨瓣和灌溉大脑表面。 </li><li>插入多柄探头，硬脑膜大带将被删除。这两个工具都需要清除硬脑膜：手术刀和昆虫针（或者，一个标准的钨电极）准备一个钩子。弯曲针尖对推动坚硬的表面（如玻璃显微镜幻灯片），它连接到一个句柄（在这里，一块木制棉条也可选择microdissecting的持针器）。 </li><li>提起钩硬脑膜，用手术刀切。采取特殊照顾，以避免损坏软，船只及大脑皮层表面。小出血可以解决生理盐水冲洗。如果出现大出血，或以任何方式损害的皮层，应考虑终止手术，并准备其他动物。 </li></ol> 6。植入探头在这个阶段，皮质表面血管的密度和方向，仔细评估。立体坐标应调整，因为探头有穿透大脑，在面积较大的船只。 植入，驱动装置可与连接立体持有鳄鱼夹举行。不间断的大脑表面的能见度和探头的秘诀是渗透成功的关键。 <ol><li>慢慢放下探头下降到约1毫米以上的预定目标，同时不断地灌溉用生理盐水开颅手术。皮层记录，探针降低到约0.5毫米的皮层和解除表面附近。 </li&gt; <li>密封用温暖融化的蜡和石蜡油混合物通过一根针（10-20G蜡石蜡油10毫升，在65°C加热）的开颅手术。申请前，混合冷却至30°C，测试密度。它应该是足够的软，便于探头运动）。为了便于全面覆盖，可熔化混合物在原地接近的微cauterizer的尖硬化蜡。 </li><li>将驱动器底部的颅骨与握水泥，小心离开螺母自由转动。在这个阶段最重要的是要避免任何意外的“凸点”的驱动，否则会损坏探头皮层。驱动器被固定在头骨后，运动平稳探头应加以核实。 </li><li>头骨水泥探头的连接部分。 </li></ol> 7。建筑上的头法拉第笼<ol><li>拉起来，组装成保护圆筒周围的探头和DRI铜网皮瓣VE。气缸也可作为电盾对环境噪声和收费行为动物晶须产生的慢波文物。 </li><li>切掉多余的材料，所以铜网与探头连接的顶部是水平调整圆柱体的高度。 </li><li>焊锡的参考和接地螺钉，导线连接器相应引脚。还焊料相邻的铜网皮瓣，以确保其电气连续性，焊接地线铜网。 </li><li>应用层上的铜网，以加强它之间的金属和动物的皮肤直接接触，以防止任何抓地力水泥。另外，申请的环氧树脂层，以进一步加强的头饰。 </li><li>测试驱动螺杆的运动。 </li><li>盖帽顶部用一块从橡胶手套切。 </li></ol> 8。在自由移动的动物录制<ol><li>后拨款吃了术后护理，录音系统，使用高阻抗探头和一个轻量级的，的ultraflexible多股电缆连接动物。抗衡的头饰的重量。 </li><li>测试的每一天记录的homecage的质量。记录点的位置来判断两个单位放电模式和局部场电位的形状。小增量（通常为1/8到1/4，每天之交，即35-70微米）的转动螺丝，直到达到目标结构，逐步降低探头。 </li></ol> 9。代表结果电生理信号（局部场电位和单位活动）取决于记录的结构和当前的动物行为。图1显示了32个通道的海马CA1区记录的例子，而老鼠是一个开放的领域探索。注意局部场电位的突出8赫兹（THETA带）在勘探与燮振荡erimposed多个柄和网站（箭头表示尖峰）扣球。分析神经元单位活动，尖峰检测和整理成单一单位使用集群分析其波形15-16。 <img src="/files/ftp_upload/3568/3568fig1.jpg" alt="图1" /> 图1。行为用4柄×8网站硅探头的大鼠海马CA1区记录。录音是宽带和采样20 000赫兹，这使得研究领域的潜力本地振荡（如“THETA”乐队的节奏8赫兹）和神经扣球活动。 <a href="http://www.jove.com/files/ftp_upload/3568/3568table1_lg.jpg"><img src="/files/ftp_upload/3568/3568table1.jpg" alt="表1" /></a> 表1。使用的试剂和设备的替代品。

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Neurosci. 8, 577-581 (2007).</li><li>Royer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-array+silicon+probes+with+integrated+optical+fibers:+light-assisted+perturbation+and+recording+of+local+neural+circuits+in+the+behaving+animal.">Multi-array silicon probes with integrated optical fibers: light-assisted perturbation and recording of local neural circuits in the behaving animal.</a> Eur. J. Neurosci. 31, 2279-2291 (2010).</li></ol>

没有利益冲突的声明。

这部影片展示了植入硅探针行为大鼠慢性大型录音程序。来自生物（脑组织）和技术（硅棒）材料的​​脆弱性的关键步骤，以确保神经元活动的质量记录。应采取特别的照顾，同时处理的探头，以避免任何接触与任何远程的“硬”的表面（例如，柄将打破，如果试图在大脑中植入他们不删除硬脑膜）柄。同样，任何脑组织的损伤（同时准备植入大脑表面，或碰撞，一旦它被植入到探针或驱动器?...

<table border="1"><tbody><tr><td> 名称 </td><td> 类型 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>硅探针Buzsaki32，4柄x 8个。包装：灵活的聚酰胺电缆</td><td>材料</td><td> NeuroNexus </td><td>探头：buzsaki32 包装：HC32 </td><td>录音探头</td></tr><tr><td>圆形的黄铜螺丝，00-90×1/2回合黄铜螺丝</td><td>材料</td><td> JIMorris </td><td> R0090B500 </td><td>传动部分</td></tr><tr><td>铜六角螺母，00-90 </td><td>材料</td><td> JIMorris </td><td> N0090B </td><td>传动部分</td></tr><tr><td>的黄铜C260地带，ASTM-B36 厚度：0.025“，长度：12”，宽度：1/2“ </td><td>材料</td><td>小零件</td><td> B000FMYU72 </td><td>传动部分</td></tr><tr><td>连接头，米，间距2毫米啤酒，单列，straigt，36个职位</td><td>材料</td><td> digikey </td><td> 2163S-36-ND </td><td>传动部分</td></tr><tr><td> 2 Sylgard硅橡胶</td><td>材料</td><td>世界精密仪器</td><td> SYLG184 </td><td>额外的绝缘探头</td></tr><tr><td> DECON Contrad 70洗衣液</td><td>试剂</td><td> Fisher Scientific则</td><td> 04-355 DECON实验室 编号：1002 </td><td>要清理的记录点</td></tr><tr><td>阻抗调理模块</td><td>设备</td><td>金控公司</td><td> 55-70-0 </td><td>阻抗计</td></tr><tr><td> niPOD - 32个通道</td><td>设备</td><td> neuronexus </td><td> niPOD -32 </td><td>阻抗计</td></tr><tr><td> “握水泥工业级</td><td>材料</td><td>填缝登士柏</td><td> 675571（粉） 675572（溶剂） </td><td>握CEMENT </td></tr><tr><td> 1,1&#39; - 十八酯-3，3,3&#39;，3&#39;-tetramethylindocarbocyanine高氯酸盐（DiI荧光“; DiIC18（3）） </td><td>试剂</td><td> Invitrogen公司</td><td> D282 </td><td>在大脑中的染色轨道探测器</td></tr><tr><td>不锈钢机械螺丝，装订头，开槽驱动，＃00-90，1/8“ </td><td>材料</td><td>小零件</td><td> MX-0090-02B </td><td>地面和参考螺丝</td></tr><tr><td>电磁线，20G，尼龙，聚氨酯涂料，MW80 </td><td>材料</td><td>小零件</td><td> B000IJYRP2 </td><td>地面和参考线</td></tr><tr><td>不锈钢机螺钉，开槽驱动绑定头部，＃000-120，1/16“ </td><td>材料</td><td>小零件</td><td> MX-000120-01B </td><td>锚螺丝</td></tr><tr><td> n-3所有目的通量液体</td><td>试剂</td><td> LA-CO（马卡尔） </td><td> 23512 </td><td>允许焊接不锈钢</td></tr><tr><td>精密微电网扩大铜</td><td>材料</td><td> dexmet </td><td> 3 CU6-050 ​​FA </td><td>铜头法拉第笼网</td></tr><tr><td>的C＆B-METABOND快！水泥体系 - 牙本质激活</td><td>材料</td><td> parkell </td><td> S380 </td><td></td></tr><tr><td>的C＆B-METABOND快！水泥体系 - 牙科水泥</td><td>材料</td><td> parkell </td><td> S380 </td><td></td></tr><tr><td>夏普点钨针和持有人</td><td>工具</td><td> roboz手术仪器</td><td> RS-6064 RS-6061 </td><td>使钩解除硬膜</td></tr><tr><td>硬质合金柏迪HP 1/4 </td><td>工具</td><td>亨利施恩</td><td> 9990013 </td><td></td></tr><tr><td>石蜡（颗粒） </td><td>材料</td><td> Fisher Scientific则</td><td> P31-500 </td><td></td></tr><tr><td>矿物油，轻（NF / FCC认证） </td><td>材料</td><td> Fisher Scientific则</td><td> O121-1 </td><td></td></tr><tr><td>气相色谱电子10-114 2部分环氧胶粘剂</td><td>材料</td><td>纽瓦克</td><td> 00Z416 </td><td></td></tr><tr><td> 1型绞合21 AWG 40/36单红聚氨酯尼龙（MW80-C），0.041“+ / -0.002”OD </td><td>材料</td><td>新英格兰电线科技有限公司</td><td> N28-36E-400-2 </td><td>使探头和放大器之间的电缆</td></tr><tr><td> 32通道超大规模集成探头，20x增益</td><td>设备</td><td> Plexon公司</td><td> HST/32V-G20 </td><td>探头</td></tr></tbody></table>

large-scale recording of neurons by movable silicon probes in behaving rodents

在神经科学中的一个主要挑战是连接到神经集会集体活动的行为。神经元和电路的输入输出关系的理解与空间的选择性和时空分辨率，适用于机械分析神经合奏动物的行为，即孤立的单个神经元代表性的大样本的录音需要的方法。乐团的神经元活动的监测显着进展在过去十年中，小型和大型脑的动物，包括人类受试者1-11。多个录制现场与以硅为基础的设备，特别是有效的，因为他们的可扩展性，体积小和几何设计。 在这里，我们描述了多个单个神经元和在啮齿动物的行为，使用市售的微加工硅探针定制的配套部件的局部场电位记录方法。有两架F基本选项或接口前置放大器的硅探针：印刷电路板和柔性电缆。探头供电公司（ <a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a> ; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a> ; <a href="http://www.acreo.se/">http://www.acreo.se/</a> ），通常会提供粘接服务和提供保税印刷电路板的探头或软电缆。在这里，我们描述了一个4柄，32现场灵活的聚酰亚胺电缆连接到探头的植入，装在一个可移动的微型硬盘。探针制备的每一步，Microdrive微型建设和手术说明，使最终用户可以很容易地复制过程。

Watch this Scientific Journal Video about Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents at JoVE.com

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

我们描述了多个单单位的大型录音和行为与硅探针的啮齿动物的局部场电位的方法。驱动器制造，探针附着到驱动器和探针植入过程容易复制足够的细节说明。

移动硅探针的神经行为鼠害的大型记录

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

large-scale-recording-neurons-movable-silicon-probes-behaving

Research

JoVE Journal

Neuroscience

34.5K 次观看.  University of New Jersey. 我们描述了多个单单位的大型录音和行为与硅探针的啮齿动物的局部场电位的方法。驱动器制造，探针附着到驱动器和探针植入过程容易复制足够的细节说明。

视频: Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

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 ...

科研

神经科学

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 ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

录音伽玛乐队在振荡脚桥核神经元

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

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

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

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

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

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

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

富氧运动器械中慢性硅探针的植入及海马位细胞的记录

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Construction of an Improved Multi-Tetrode Hyperdrive for Large-Scale Neural Recording in Behaving Rats

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems neuroscience. One key component of this technique consists of the precise placement of multiple electrodes into desired brain regions and the maintenance of their stability. Here, we describe a protocol for the construction of a 3D-printable hyperdrive, which includes eighteen independently adjustable tetrodes, and is specifically designed for in vivo extracellular neural recording in freely behaving rats. The tetrodes attached to the microdrives can either be individually advanced into multiple brain regions along the track, or can be used to place an array of electrodes into a smaller area. The multiple tetrodes allow for simultaneous examination of action potentials from dozens of individual neurons, as well as local field potentials from populations of neurons in the brain during active behavior. In addition, the design provides for simpler 3D drafting software that can easily be modified for differing experimental needs.

We present the construction of a 3D-printable hyperdrive with eighteen independently adjustable tetrodes. The hyperdrive is designed to record brain activity in freely behaving rats over a period of several weeks.

一种改进的多电子四极超光速引擎在行为大鼠大尺度神经记录中的构建

Monitoring the activity patterns of a large population of neurons over many days in awake animals is a valuable technique in the field of systems ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...