作者感谢尼克·凯斯曼的建议，并在准备稿件的帮助。这种技术是与由AFOSR下批FA9550-10-1-0054，并根据批准号:IOS-1120305美国国家科学基金会支持的快线RER工作联合开发。

1。四极管导线的制备<ol><li>拉出约1.1米长的一个非常薄的镍铬合金丝（12微米直径，PAC涂层）。附加的磁带标签，每一端。挂线在一个水平螺纹杆，使得两个端部在靠近台面相同的高度。 </li><li>重复步骤1.1为第二导线，使得总共4 2多个端部，并把它旁边的第一金属丝（约1厘米之间）。 </li><li>用胶带粘标签的四个两端连在一起，并在标签附加到一个电动旋转卷取装置。这种装置可以由廉价的直流电机进行。 </li><li>缠绕四极管在一个方向上持续2分钟（每分钟60转）和放松它在相反方向上，持续30秒。 </li><li>使用热风枪，融合在一起的电线。不要触摸电线枪。使用三个向上和向下传递的交替方向，每个通取约10秒。 </li><li>切割顶部和伤口线的底部。把四根导线绞合的ð融合在一起的一端分开，但在其他。 </li><li>新增配​​套管。切聚乙烯管材有30厘米长（直径:内0.28毫米，外0.61毫米）。线程四极管非常缓慢和谨慎到支撑管，使其不纠结。 </li><li>一旦融合的端部出现了另一侧，通过拉它，以便有金属丝的位于所述导向管的两端部的长度相等。 </li><li>抓住每根线用钳子的独立结束。使用气体燃烧器的火焰的基部，小心地燃烧的绝缘关闭的最后2或3mm的每个导线。加热丝，直到它发光，但不卷曲。 </li><li>连接与四极适合您的记录装置的男女IC插座适配器。把每根线的deinsulated端插入不同的插座用钳适配器。稳定线与一个小铜针插座。用细一点的烙铁，并填写插座与熔化的焊料。注意不要接触脆弱的金属丝用烙铁。 </li><li>检查每根电缆的阻抗与各对导线的跨阻抗。 <ol><li>将熔融的，扭曲的一端插入生理盐水的容器中，从盐的铜导线导体连接到欧姆计。 </li><li>连接米到含有金属丝的插座针的另一端。每根导线的阻抗应小于3MΩ。 </li><li>如果上述值不达到，重新尝试焊料连接。 </li><li>从盐水中取出电线，用清水冲洗干净的提示，并测试跨线阻抗为每个配对（N = 6）。跨阻抗应大于5MΩ。 </li><li>如果上面的值没有达到，切片少量尖断的融合结束并重新测试。 </li><li>丢弃任何线组不符合双方的所有导线的阻抗要求。 </li></ol></li><li>确保四极管。 <ol><li>折叠一个长方形的小纸盒slightly比插座适配器大。 </li><li>适配器转成箱的男性侧面底部。渗透使得男性侧的所有引脚框外侧，而适配器的其余部分是箱内箱。 </li><li>磁带上的​​外箱的角上。使用在盒子的内部小块双面胶带，以稳定金属丝的任何单个线材。在退出方框中的线应熔合。 </li><li>混合快速设置2部分环氧树脂倒入框固定适配器和所有的电线。 </li><li>附加导油管的近端与牙模框的一侧，但离开管道打开，使得四极管，可以通过自由两端拉。 </li></ol></li><li>锐化的四极管。 <ol><li>每次实验之前，切四极管的前端用锋利的解剖刀刀片，而不是剪刀。这可以防止破碎和同时提供一个干净的平面边缘的下一个步骤中的金属丝的张开结束。 <oL&gt; <li>使用小旋转刀具垂直安装中，细砂砾打磨盘（这​​些都可以在一个平台上进行组合）来打磨四极管和删除一些尖端绝缘。握住束近其与镊子结束。倾斜线设置结束45°角相对于打磨盘，轻轻地触摸到中速旋转盘约1或2秒每次在媒体上，然后细糁。重复此三次，每次轴向旋转的捆90°。至关重要的是，在打磨盘的旋转的方向是远离导线端部的小角度，否则可能发生的电线分离。 </li><li>所期望的结果变换束一端从直边到尖与每根电线的端部除去少量的绝缘层。使用解剖显微镜电镀四极管前验证这一点。如果发生在尖端任何磨损，重新切割和重新抛光。 </li><li>如果subsequ期间阻抗测试耳鼻喉科电镀步骤显示了极低的跨线值（小于4MΩ），它表示在抛光步骤去除过多的材料。重新切割和重新抛光的四极管。 </li></ol></li><li>板的四极管。把四极管的前端成饱和硫酸铜溶液（85毫升水中，加入5ml硫酸50克硫酸铜）。板每根导线与2.5μA与刺激隔离器的电流。注入电流为1秒，停顿1秒，然后重复这个过程4倍。 </li><li>检查每根电缆的阻抗和每一对导线的interimpedance。每根导线的阻抗应0.5-1MΩ和跨阻抗应大于4MΩ之间。 </li><li>安装适配器到多声道录音系统的探头。 </li><li>附加弯曲昆虫针显微操作。四极管的尖端附着在昆虫针牙科蜡</li></ol></li></ol> 2。动物的制备<ol><li>麻醉蟑螂用冰冷却。 </li><li>之后蟑螂停止移动，垂直抑制蟑螂在平坦的软木表面有大鞍销跨越昆虫，但不穿透任何部分它的身体。 </li><li>制备转移至塑料容器中，并放置冰上周围的动物，以尽量减少血液流动和身体动作。 </li><li>放置一个塑料套在脖子上，以支撑头部，然后将牙模的头部周围以使其稳定。 </li><li>切单眼之间有一个小窗口用刀片从头部取出的角质层。 </li><li>去除结缔组织和脂肪用钳，露出大脑。 </li><li>放置一些蟑螂盐水到头部胶囊以覆盖脑组织。 </li><li>要desheath大脑，用细镊子轻轻抓住对大脑的顶部外皮和使用其他细镊子撕鞘除了在电线植入区域。 </li><li>开一个小口子头壳前到大脑的智慧ħ昆虫针。插入的绝缘铜导线插入孔作为参考/接地电极三个较大的直径（56微米）编织。 </li><li>四极管的尖端降低到大脑表面的显微操纵器和它附近感兴趣的大脑区域的位置。 </li><li>小心地将两小片薄醋酸酯片（2毫米x 1毫米），比头壳孔略大，前部和后部的四极管。 </li><li>把该记录系统上。 </li><li>慢慢放下四极150-250微米的大脑表面下视录制质量。 </li><li>关闭记录系统。 </li><li>将两片醋酸片尽可能靠近四极管尽可能不触及它（ 图1A）。 </li><li>发热量小抹刀或扁平的皮下注射针头，放入牙蜡，有液体蜡锅铲一角。仔细触摸每一片片醋酸从远端的用刮刀式四极管，使液体蜡可以流到每片和密封它与头部表皮之间的差距。 </li><li>重复步骤2.16。滴少量液体蜡到每次乙纸。启动过程远离四极管，并逐步向移动。最终，四极管将牙模被锚定。避免让热蜡进入型腔，并到大脑。 </li><li>使用同样的方法，步骤2.16和2.17的锚用蜡参考/接地电极。 </li><li>加热，重视四极管的显微操纵器，从它释放四极蜡。 </li><li>环四极管到牙模的头部提供了应力消除（ 图1B）。 </li><li>盖应力消除环与牙模（ 图1C）。 </li><li>小心地取下约束和准备转移到培养皿中。大马鞍针抑制编制背侧。 </li><li>附使用胶枪一杆到前胸背板。这是一个木棍从前胸背板在腹部延伸。 </li><li>四极管的前端连接到用牙科用蜡在杆的后端。 </li><li>锚定四极管和参考/接地电极，以与牙科用蜡棒的前端。 </li><li>从管道尽可能的插座端拉四极管，但不用力拉它，为了消除的可能性，该动物可能油管（ 图1D）以外损坏四极管的部分。 </li><li>卸下所有的约束。附上参考/接地电极的四极管，带牙模。 </li><li>等待至少60分钟的动物从任何实验前冰麻醉中恢复过来。 </li></ol> 3。实验方法<ol><li>连接一台PC同时与记录系统，并使用USB转串口线的LED灯。 </li><li>神经开始录音。 </li><li>开始录像，每秒20帧的使用，使用高速摄像机爬坡实验Motmot图像采集包16或120 fps的行走实验。 </li><li>将蟑螂进入低40厘米×40厘米的有机玻璃舞台上行走实验或58厘米长5厘米宽，5厘米高的舞台上攀登实验。行走江湖有从右侧墙壁中间延伸到舞台，高于该探头是位于市中心的透明屏障。屏障是用来防止动物行走在摄像机视野受阻的探头的区域。登山舞台拥有丙烯酸块（无论是1.2厘米或1.8厘米高，5厘米宽），或者位于一个可比的高度在中心的架子上。 </li><li>生成从PC使用一个自定义的MATLAB命令TTL脉冲。 （S =串行（&#39;COM4&#39;）;打开（次）; s.RequestToSend =&#39;关闭&#39;/ s.RequestToSend =&#39;上&#39;/; FCLOSE（次），删除（S）;)。该TTL脉冲产生一个次夯实记录系统，要么打开或关闭LED灯。 </li><li>让蟑螂探索舞台，直到它停止移动超过30秒，行走实验。让蟑螂要么翻越块/架或隧道穿过货架攀登实验。 </li><li>停止录像。 </li><li>神经停止录音。 </li><li>写下来的TTL脉冲产生的时间戳。 </li><li>从舞台上移除蟑螂，并等待至少3分钟。 </li><li>下一个试验重复步骤3.2-3.10。 </li><li>一旦所有的记录已完成，经过5秒5μA直流电流通过的金属线尖端（阳极）和参考电极（阴极）以沉积铜进入大脑的金属丝端头之一。 </li></ol> 4。离线分析<ol><li>通过其中的LED灯被切换的帧，并在由记录系统的时间戳联的视频和神经数据同步那一刻。 </li><li>马克丝尖端位置。使用蒂姆斯集约化过程中沉淀，观察铜在12微米的连续切片17。突出存款应在3-8相邻部分（大约18-48％的区域的背腹平面的长度的，我们从记录）（ 图2）是可见的。 </li><li>相关具体电脉冲单个神经元的活动。按照秒杀奠定了在其他地方详细10,14,18排序程序。使用该程序KlustaKwik（1.5版，作者K.哈里斯，罗格斯大学）生成初始，自动聚类。将它们导入到程序MClust（3.5版明尼苏达大学，公元作者瑞迪等 ，）的进一步细化和分析（ 图3）。 </li><li>跟踪蟑螂的动作。行走实验中，提取蟑螂（视觉）中的VID的每一帧的质量和它的身体取向的中心的位置和相关的FixErrors的MATLAB工具箱19;使用加州理工学院的多个飞跟踪（http://ctrax.sourceforge.net/ 0.1.5.6版本）EO录音。攀登实验，提取该块和蟑螂的头部和前胸背板中使用运动分析软件包的视频的每个帧中的位置。 </li></ol>

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作者宣称没有利益冲突。

而对昆虫大脑的CC或其他地区以前的电生理研究为我们提供了见解行为的控制中枢，他们大多在任内敛准备9,11或拴那些10,14分别进行。其结果是，在动物的感官体验和生理状态可以是从那些在自然的环境非常不同。此外，这种动物可以执行行为的任务是有限的，以一个平面下的那些情况。在这里，我们提出了一个方法，从自由活动的蟑螂消委会记录。但愿，我们已经为您提供了所?...

本文介绍了一个成功的系统，从内部的中央复杂的蟑螂，Blaberus discoidalis的（CC）的神经元记录，如昆虫走在舞台上，并处理导致它转身，隧道下，或翻越障碍的对象。导线也可以连接到一个激励器，以唤起活性在周围神经纤维与随后的行为变化。 在过去的十年中相当大的关注已经直指在控制昆虫行为的不同脑区所扮演的角色。这大部分的焦点已经指向被统称为中央复合体（CC）中线脑neuropils。已经取得进展，作为种类繁多的目标有关的行为对CC的作用问题技术的结果。这些技术包括从神经源性的操作，主要是在果蝇中 ，加上behavi口服分析1-3，该监测CC和尝试与该活动相关的行为上的参数范围内的神经活动电生理技术。 电生理技术，包括细胞内记录的个人识别4-9的神经元和细胞外记录，常与多通道探头10,11。这两种技术都是免费的。细胞内记录用锋利的电极或全细胞膜片提供鉴定神经元非常详细的数据，但仅限于一个或两个细胞一次，需要限制或无运动，能维持的时间相对较短。细胞外记录可以很容易地设置，不要求限制，并且可以保持几个小时。随着多通道四极管和集群切割，神经元的相当大的群体可以同时9,12分析。而整个细胞PATCH有栓系昆虫13被成功地使用，我们觉得也有必要的技术，使我们能够在大脑中记录神经活动的时间在自由活动的昆虫很长时间，因为他们对付障碍向前运动。 记录作为昆虫的动作和反弹向上和向下的需要推动我们朝着细胞外记录的方法。我们有良好的成功记录在内敛的准备与市售的16个通道的硅探针11，但即使是大蟑螂的小尺寸意味着该探测器必须安装脱体。也就是说，加上探针尖头的美味，使他们不适合免费步行的准备。在前面两个项目中，我们使用的细线形成四极管来完成类似的记录特性捆绑，但在一个更强大的安排。这些四极束使我们能够从束缚蟑螂录制ð消委会有关单位活动的变化，行走速度14和转向从一个杆10触角接触产生的行为。 很有用，因为这些束缚准备一直并将继续是，他们的确存在一定的局限性。首先，该昆虫可以执行的行为是仅限于一个平面上。也就是说，我们可以很容易唤起改变行走速度或转弯，但登山和隧道的行动是不可能的，至少具有典型的系绳安排。其次，我们的系留的准备工作"开环"。也就是说，它们不允许正常运动相关的反馈的系统。因此，当蟑螂开启我们的系绳，其视觉世界并没有相应地改变。这是不可能建立闭环系绳系统引入这种反馈。然而，它们是由模拟的视觉环境的编程和硬件的复杂性的限制。 Nevertheles秒，我们觉得我们可以改善我们现有拴记录方法从动物记录，因为它在舞台上或轨道和遇到的对象，因为它会在其自然环境自由地走了。 虽然无线系统，用于记录大脑活动15将是理想的，当前的系统中有记录的信道数，数据采集，电池寿命和重量的时间限制。因此，我们选择了尽量去适应我们的使用系留记录系统中自由移动的准备工作。为更好的无线系统变得可用，这种技术可以很容易地适应于这样的设备。本文中所描述的系统具有重量轻，工作得很好，似乎对蟑螂的行为有点不利的影响。与廉价的高速摄像头和簇切割软件，在个别大脑神经元的活动可能与运动。在这里，我们描述了prepar四极导线和植入昆虫的大脑，以及录音技术的电活动和运动，以及这些数据的振动性可汇聚供后续分析。

<table border="0" cellpadding="0" cellspacing="0" width="728">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:207px;">Nichrome wire&#160;</td>
			<td style="width:179px;">Sandvik Heating Technology</td>
			<td style="width:137px;">Kanthal RO-800</td>
			<td style="width:205px;">Use for tetrode</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biomedical polyethylene tubing</td>
			<td>A-M Systems</td>
			<td>800700</td>
			<td>Use for tetrode tubing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lynx-8</td>
			<td>Neuralynx</td>
			<td></td>
			<td>Use for multi-unit recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cheetah 32</td>
			<td>Neuralynx</td>
			<td></td>
			<td>Use for multi-unit recording</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High speed camera</td>
			<td>Basler</td>
			<td>A602f</td>
			<td>Use fir video recording for walking experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High speed camera</td>
			<td>Casio</td>
			<td>EX-FC150&#160;</td>
			<td>Use for video recording for climbing experiments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WINanalyze</td>
			<td>Winanalyze</td>
			<td>version 1.4 3D</td>
			<td>Use for video tracking&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matlab</td>
			<td>MathWorks</td>
			<td>MATLAB R2012b</td>
			<td>Use for TTL pulse generation and off-line data analysis</td>
		</tr>
	</tbody>
</table>

extracellular wire tetrode recording in brain of freely walking insects

大脑活动的昆虫电机控制中的作用越来越大的兴趣，需要，我们能够监视的神经活动，而昆虫进行自然的行为。我们以前开发的植入四极导线插入蟑螂的大脑，使我们能够从多个记录的神经元同时活动的中央复杂的技术，而拴蟑螂开启或改变行走速度。而一大进步，拴准备提供访问有限的行为，且往往缺乏发生在自由活动动物的反馈过程。我们现在提出的技术，使我们能够从自由移动蟑螂，因为他们走在舞台上，并通过旋转，爬坡或隧道处理障碍中心的复杂记录的修改版本。再加上高速视频和集群切割，我们现在可以与大脑活动的自由活动的昆虫运动的各种参数。

我们从27准备步行实验消委会录得50个单位的神经活动。 15那些准备（23单位），还进行攀岩实验。单个的单元是按照制剂和单元编号命名（ 例如，单元1-2表示制剂1，单元2）。 一个爬坡试验的视频快照， 如图4所示，整个视频可在补充视频1（声音从单元1-2）。录音是在正确的扇形体（FB）。蟑螂停下脚步，当它遇到的块，并用它的...

Watch this Scientific Journal Video about Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects at JoVE.com

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

我们以前开发的植入四极导线插入蟑螂大脑的中央复杂，使我们能够监视活动在拴蟑螂个别单位的技术。在这里，我们提出的技术，使我们能够也可自由移动的昆虫记录大脑活动的修改版本。

外导线四极管记录在行走自如昆虫大脑

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform ...

extracellular-wire-tetrode-recording-brain-freely-walking

Research

JoVE Journal

Neuroscience

16.5K 次观看.  Case Western Reserve University. 我们以前开发的植入四极导线插入蟑螂大脑的中央复杂，使我们能够监视活动在拴蟑螂个别单位的技术。在这里，我们提出的技术，使我们能够也可自由移动的昆虫记录大脑活动的修改版本。 

视频: Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

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

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

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

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

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

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

科研

神经科学

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

在完整的成年斑马鱼大脑电生理记录

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Recording Single Neurons' Action Potentials from Freely Moving Pigeons Across Three Stages of Learning

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed single-neuron activity while animals are acquiring an operantly conditioned response, or when that response is extinguished. But even in these cases, observation periods usually encompass only a single stage of learning, i.e. acquisition or extinction, but not both (exceptions include protocols employing reversal learning; see Bingman&#160;et al.1 for an example). However, acquisition and extinction entail different learning mechanisms and are therefore expected to be accompanied by different types and/or loci of neural plasticity.
Accordingly, we developed a behavioral paradigm which institutes three stages of learning in a single behavioral session and which is well suited for the simultaneous recording of single neurons&#39; action potentials. Animals are trained on a single-interval forced choice task which requires mapping each of two possible choice responses to the presentation of different novel visual stimuli (acquisition). After having reached a predefined performance criterion, one of the two choice responses is no longer reinforced (extinction). Following a certain decrement in performance level, correct responses are reinforced again (reacquisition). By using a new set of stimuli in every session, animals can undergo the acquisition-extinction-reacquisition process repeatedly. Because all three stages of learning occur in a single behavioral session, the paradigm is ideal for the simultaneous observation of the spiking output of multiple single neurons. We use pigeons as model systems, but the task can easily be adapted to any other species capable of conditioned discrimination learning.

Learning new stimulus-response associations engages a wide range of neural processes which are ultimately reflected in changing spike output of individual neurons. Here we describe a behavioral protocol allowing for the continuous registration of single-neuron activity while animals acquire, extinguish, and reacquire a conditioned response within a single experimental session.

自由移动鸽子穿越三个阶段学习记录单个神经元&#39;动作电位

While the subject of learning has attracted immense interest from both behavioral and neural scientists, only relatively few investigators have observed ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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 ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

鼠脑切片偶联微电极阵列癫痫活性的记录与调控

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...

Hybrid Microdrive System with Recoverable Opto-Silicon Probe and Tetrode for Dual-Site High Density Recording in Freely Moving Mice

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, conventional microdrive designs often only allow use of one type of electrode to record from single or multiple regions, limiting the yield of single-unit or depth profile recordings. It also often limits the ability to combine electrode recordings with optogenetic tools to target pathway and/or cell type specific activity. Presented here is a hybrid microdrive array for freely moving mice to optimize yield and a description of its fabrication and reuse of the microdrive array. The current design employs nine tetrodes and one opto-silicon probe implanted in two different brain areas simultaneously in freely moving mice. The tetrodes and the opto-silicon probe are independently adjustable along the dorsoventral axis in the brain to maximize the yield of unit and oscillatory activities. This microdrive array also incorporates a set-up for light, mediating optogenetic manipulation to investigate the regional- or cell type-specific responses and functions of long-range neural circuits. In addition, the opto-silicon probe can be safely recovered and reused after each experiment. Because the microdrive array consists of 3D-printed parts, the design of microdrives can be easily modified to accommodate various settings. First described is the design of the microdrive array and how to attach the optical fiber to a silicon probe for optogenetics experiments, followed by fabrication of the tetrode bundle and implantation of the array into a mouse brain. The recording of local field potentials and unit spiking combined with optogenetic stimulation also demonstrate feasibility of the microdrive array system in freely moving mice.

This protocol describes the construction of a hybrid microdrive array that allows implantation of nine independently adjustable tetrodes and one adjustable opto-silicon probe in two brain regions in freely moving mice. Also demonstrated is a method for safely recovering and reusing the opto-silicon probe for multiple purposes.

混合微驱系统,带可回收的光电探头和Tetrode,用于自由移动小鼠的双位点高密度记录

Multi-regional neural recordings can provide crucial information to understanding fine-timescale interactions between multiple brain regions. However, ...

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

多纤维光测量记录自由移动动物的神经活动

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...