这项工作得到了韩国科学和技术研究所项目 (No. 2E26190 和 2E26170) 和人类前沿科学项目 (RGY0089/2012) 的支持。

所描述的所有方法均已得到韩国科技研究院动物保育和使用委员会的批准. 
 1. 准备微驱动器和电极 <ol> <li> 组装微驱动器. 
	<ol> <li> 使用高分辨率3D 打印机打印微驱动器 (滑块、正文和外壳) 14 的各个部分。确保零件没有缺陷. </li>
		<li> 将滑块固定到带有螺钉的微驱动器体 (大小 000-120x1/4). </li>
		<li> 将螺母 (大小 000-120x5/64 十六进制) 焊接到螺钉的顶端. </li>
		<li> 顺时针旋转螺钉以将滑块向上或逆时针移动以将其向下移动. </li>
		<li> 用螺钉将外壳固定在机身上 (大小 000-120x1/4). </li>
	</ol> </li> <li> 在微驱动器上安装探测器。
	<ol> <li> 在双目显微镜下将微驱动器固定在水平位置. </li>
		<li> 使用带橡胶填料的扁平鳄鱼夹来抓取硅探针的挠性电缆, 同时小心地将探头从装运箱中分离出来, 用镊子。将鳄鱼夹子固定在机械手上. </li>
		<li> 使用机械手, 将硅探针放置在微驱动滑块上, 与移动方向平行. </li>
		<li> 应用一滴牙科水泥 (室温固化丙烯酸牙科修复材料) 将探头固定在滑块上。如果探头位置已移位, 请更正它。胶水是不推荐的, 因为它的治疗非常快, 使它很难重新调整电极的位置. </li>
		<li> 将探头连接器固定在带有牙科水泥的 C 型支架 (连接器支架) 上. </li>
	</ol> </li> <li> 清洗并将染料置于探头上. 
	<ol> <li> 修复探针清洗设备上的微/探头组合。该装置配备2小旋转泡沫海绵 (消毒拭子)。用机械手调节间隙. </li>
		<li> 用洗涤剂浸泡海绵. </li>
		<li> 慢慢地在海绵之间移动探头, 允许轻轻的触摸。在显微镜下监控清洗过程. </li>
		<li> 用蒸馏水冲洗探头。将探头浸入酒精中进行灭菌. </li>
		<li> 用泡沫拭子将 Dil (亲油性荧光染料) 涂抹在探针的柄的背面。这将允许在稍后阶段的可视化小腿位置在大脑中. </li>
	</ol> </li> <li> 组装 hat <ol> <li> 使用 3 d 打印机打印帽子 (头板、连接器和 cap) 14 的各个部分。3零件合在一起形成一个密封的外壳. </li>
		<li> 在磁头板的插槽中插入螺母 (大小 M2 和 00-90x1/4), 并用胶水和牙科水泥固定. </li>
		<li> 在瓶盖的插槽中插入垫圈并修复牙科水泥. </li>
	</ol> </li> </ol> 2。颅骨固定的手术方法 所有用于手术的工具都事先由灭菌消毒。干热珠装置是使用 forto 消毒工具, 成为污染, 并需要在手术中消毒. 
<ol> <li> 将异氟醚的水平设置为麻醉开始时的4%。将鼠标放在麻醉室中5分钟. </li>
	<li> 将鼠标安装在立体定向设备中. </li>
	<li> 将异氟烷水平降低到 1.5-2%。根据动物状态、呼吸速率和体温 20 调整手术的水平. </li>
	<li> 在眼睛上涂抹眼药膏. </li>
	<li> 用防腐剂 (iodopovidone) 将头皮剃光并清洁动物头部。在手术的所有步骤中保持无菌状态. </li>
	<li> 在头皮下注射布比卡因 (1 毫克/千克)。用细剪刀切开和移除小鼠头部的顶骨部分, 露出头骨的边缘。在手术中使用生理盐水和止血海绵清洗和控制出血. </li>
	<li> 使用刮刀工具拆卸骨膜. </li>
	<li> 查找头骨上的参考点 21 : bregma、λ、冠状和矢状缝合线。调整头部和 #39 的角度沿矢状轴, 使 Bregma 和 lambda 点处于同一高度. </li>
	<li> 在头骨上钻两个孔 (直径为0.5 毫米), 用于参考和接地电极。孔应该是大约1毫米尾鳍和1毫米侧向λ. </li>
	<li> 插入地面和参考电极 (尺寸 000-120x1/16 微型不锈钢螺丝线-耦合到针连接器). </li>
	<li> 在头骨上应用紫外线 (uv) 光合牙本质活化剂, 然后在45-60 秒应用 uv 光. </li>
	<li> 在头骨的边缘应用一层牙科水泥。避免在老鼠的皮肤和毛发上涂上水泥. </li>
	<li> 将磁头板固定在一个立体定位机械手上, 并将其置于颅骨上方。慢慢降低头部板, 直到它稍微触及头骨, 并应用牙科水泥在与头骨的交界处。让牙科水泥固化15分钟. </li>
	<li> 删除麻醉。将连接器和帽固定在头板上。在给 sub-cutaneous 注射 ketaprofen 5 毫克/千克后, 把老鼠放在笼子里. </li>
	<li> 给 sub-cutaneous 注射 ketaprofen 5 毫克/千克的两天, 并仔细监测任何疼痛的迹象。小鼠通常在20-40 分钟内从麻醉中苏醒。这种帽子的植入物相对较轻 (3.34g), 这样老鼠就不会有麻烦抬起头, 在迷宫中奔跑, 爬到他们家笼子的边缘. </li>
</ol> 3。行为训练 <ol> <li> 在后恢复期为7天后, 开始限制每天1毫升的水. </li>
	<li> 制作跑步机皮带, 切一块天鹅绒面料 (5 厘米乘1-2 米), 用热胶固定小物件。在皮带的边缘附加竖立的物体, 以免干扰鼠标的运动. </li>
	<li> 将两个端点缝合在一起, 固定在跑步机轮子上的皮带. </li>
	<li> 将磁头板的两个螺钉插入并拧紧到磁头固定板的插槽中, 将鼠标固定在跑步机上. </li>
	<li> 开始训练鼠标在跑步机上运行头部限制, 以获得水奖励。通过舔口提供水的奖励。最初, 把鼠标放在跑步机上10分钟, 每天3次. </li>
	<li> 当鼠标习惯于头部固定并开始移动皮带 (通常在3天后), 将训练会话持续时间增加到30分钟。2-3 周后, 一些老鼠在30分钟内跑了超过100次试验 (试验是皮带的完全旋转). </li>
	<li> 选择显示记录实验最佳行为表现的小鼠. </li>
</ol> 4。植入硅探针 <ol> <li> 将鼠标置于麻醉下. </li>
	<li> 通过固定头板将鼠标安装在立体定向装置中。用盐水清洗颅骨表面. </li>
	<li> 查找立体定向标记: bregma、λ、冠状和矢状缝合。测量到插入点的距离并标记它. </li>
	<li> 仔细地钻骨, 直到它变得薄而透明。在钻孔时用盐水滋润和清洁. </li>
	<li> 使用精确的力, 小心地去除薄骨和硬脑膜物质所有的时间保持脑表面湿润. </li>
	<li> 将微/硅探针组合固定在一个立体定位机械手上。把硅探针放在开颅上面将硅探针连接器固定在磁头板上. </li>
	<li> 连接记录放大器和接地/参考电极。用铝箔屏蔽鼠标以防止电磁噪声。启动记录系统以监视大脑的电活动. </li>
	<li> 使用微缓慢地将硅探针插入大脑。不断检查电信号, 机械手走过的距离, 和探针的小腿 (确保他们在大脑中穿透)。单位活动是可见的皮层, 而下方的白色物质是相对沉默。当小腿触及海马的锥体层时, 单位活动就会重现。从这一点上, 收回硅探针200和 #181; m (使用微驱动器的第二天, 使小腿回到金字塔层). </li>
	<li> 用骨蜡和矿物油的混合物覆盖大脑表面. </li>
	<li> 使用牙科水泥将微驱动器固定到磁头板上。让牙科水泥固化15分钟。然后将微驱动器从立体定向机械手中分离出来, 再戴上帽子帽. </li>
	<li> 将鼠标放回笼中, 并给出 ketaprofen 5 毫克/千克的 sub-cutaneous 注射。检查任何疼痛的迹象。这种手术比第一个更少的侵入和小鼠通常活跃在45分钟后, 他们从麻醉唤醒。然而, 当硅探针需要在大脑中稳定时, 让老鼠恢复一整天. </li>
</ol> 5。录制 <ol> <li> 在跑步机上安装鼠标头部固定。取下帽帽. </li>
	<li> 连接录音放大器并启动录制. </li>
	<li> 在第一天, 使用微驱动器将硅探针移动到海马的锥体层。每个逆时针旋转的螺丝移动小腿200和 #181; 我更深。在出现波纹振荡 13 和设备活动之前, 慢慢调整刀柄位置 (20-50 和 #181; m). </li>
	<li> 在第二天, 调整小腿位置和等待〜1小时之前记录海马活动, 而鼠标在皮带上运行。为了保持良好的记录信号质量几天, 删除硬物体和低天花板, 从鼠标和 #39; s 笼, 以尽量减少的机会, 帽子颠簸到坚硬的表面. </li>
</ol> 6。正在恢复探测器 <ol> <li> 将鼠标置于麻醉下. </li>
	<li> 通过固定头板将鼠标安装在立体定向装置中。取下帽帽. </li>
	<li> 将微驱动器上的立体定向机械手带上。将微驱动器固定在机械手上。从磁头板上拧下接头支架。卸下连接微型驱动器外壳和车身的螺钉. </li>
	<li> 在双目显微镜监视下, 用立体定向机械手缓慢拉动微驱动器/硅探针组合, 使壳体部分落后. </li>
	<li> 使用清洗设备清洗硅探针. </li>
</ol>

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+Contribution+of+Adult-Born+Hippocampal+Granule+Cells+to+Context+Encoding.">Distinct Contribution of Adult-Born Hippocampal Granule Cells to Context Encoding.</a> Neuron. 90 (1), 101-112 (2016).</li><li>Stark, E., Koos, T., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diode+probes+for+spatiotemporal+optical+control+of+multiple+neurons+in+freely+moving+animals.">Diode probes for spatiotemporal optical control of multiple neurons in freely moving animals.</a> J Neurophysiol. 108 (1), 349-363 (2012).</li><li>Wu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+neural+probe+with+monolithically+integrated+dielectric+waveguide+and+recording+electrodes+for+optogenetics+applications.">An implantable neural probe with monolithically integrated dielectric waveguide and recording electrodes for optogenetics applications.</a> J Neural Eng. 10 (5), 056012 (2013).</li></ol>

作者没有什么可透露的。

慢性记录的神经元活动是关键的了解神经过程, 如海马位场。我们的方法来执行慢性硅探针 implantantation 区别自己从其他方法7,18,19,20的事实是, 它是相对简单的恢复电极封装在实验的结束。虽然地方细胞通常是在自由移动的条件下进行研究的, 但跑步机设备不仅能极大地简化实验设计和数据分析, 还可以让研究人员在最小的环境中仔细地观察场地机制许多重复套用老调动物轨道12。它也比虚拟现实系统更简单, 因为它只需要3维打印车轮, one-dimensional 运动传感器, 和单片机。
稳定的场地活动可以在许多天内记录下来, 这是调查与学习有关的长期网络动态的重要条件。在这方面, 应考虑一些问题。首先, 硅胶探针周围的脑组织变性和瘢痕形成的过程本身可能会产生长期的活动模式变化。因此, 所有的外科手术都要小心, 以便对脑组织和血管造成最小的损伤。其次, 由于电极在大脑中的漂移14, 在连续的天数内跟踪单个细胞相对困难。在这方面, 像双光子显微镜和微内窥镜这样的成像技术可能更合适, 因为细胞的特征可以从细胞的形态和空间结构中追踪21,22,23.它们还允许在记录的细胞数量方面获得更大的收益, 并提供有关细胞基因表达式的直接信息24。然而, 成像技术是相当侵入时, 深脑区的关注, 不能解决单一的动作电位, 因为他们主要依靠钙信号。因此, 纵向研究的最佳解决方案可能是结合光学和电生理方法。
最后, 值得注意的是, 我们所描述的技术集可能很容易适应其他类型的电极, 包括 Neuropixels (https://www.janelia.org/lab/harris-lab-apig) 和 optrodes11,25,26, 可用于除海马位细胞以外的一系列实验模型。随着 3 d 印刷技术的飞速发展, 对记录和行为设备的进一步改进和定制应该变得越来越直接和容易进入实验室。

硅探针为电生理记录提供了几个好处, 包括事实他们是设计的以锋利的外形最小化组织损伤, 并且他们提出一个密集地被包装的录音站点的一个精确布局1, 2、3、4。它们用于研究不同物种的各种系统, 包括人类3,5,6, 具有不同的方法1,7。然而, 由于它们的成本、脆弱性以及慢性实验的简便方法缺乏8, 它们的经常性使用仍然相对有限。在3D 印刷技术的最新进展使定制设计的设备, 如微型驱动器和头部板, 使更容易处理这些微妙的电极。在第一步中, 我们将描述如何构建和使用我们为植入慢性硅探针而开发的一套工具14。
虽然地方细胞通常是研究使用自由移动的动物在迷宫中运行, 最近他们也在虚拟环境中进行了15和在跑步机 apparatii9 (图 1A) 中进行了调查。这些实验方法提供的优势, 动物可以头部克制, 使使用2光子显微镜15, 补丁钳16, 和传感9,10,11技术更容易, 除了提供增强的控制动物行为和环境提示12。在第二步中, 我们将介绍训练小鼠和记录在跑步机设备中的细胞活动的程序。

<table>
	<tbody>
		<tr>
			<td>Silicon Probe</td>
			<td>Neuronexus</td>
			<td>Buzsabi32</td>
			<td>Recording electrode</td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Intantech</td>
			<td>RHD2132/RHD2000</td>
			<td></td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Asiga</td>
			<td>Pico Plus 27</td>
			<td>High resolution printer for micro-drive</td>
		</tr>
		<tr>
			<td>3D printer</td>
			<td>Stratasys</td>
			<td>Mojo</td>
			<td>Lower resolution printer for hat components</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>Kopf</td>
			<td>Model 963</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Treadmill apparatus</td>
			<td></td>
			<td></td>
			<td>We build them</td>
		</tr>
	</tbody>
</table>

implantation of chronic silicon probes and recording of hippocampal place cells in an enriched treadmill apparatus

了解大脑功能的一个重要条件是识别行为和细胞活性的相关性。硅探针是用于 large-scale 神经元活动的电生理记录的高级电极, 但其慢性植入的程序仍然不发达。已知海马位细胞的活动与动物在环境中的位置有关, 但其基本机制尚不清楚。为了研究地方细胞, 我们在这里描述了一系列的技术, 从制造设备的慢性硅探针植入到监测的地方现场活动在一个提示丰富的跑步机设备。一个微型驱动和帽子是由装配和紧固在一起 3 d 打印塑料零件。硅探针安装在微驱动器上, 清洗, 并涂上染料。第一次手术是为了修复老鼠头骨上的帽子。小地标是捏造的, 并附着在跑步机的皮带上。老鼠被训练在跑步机上运行头部固定。第二个手术是在海马内植入硅探针, 然后记录下宽带电生理信号。最后, 对硅探针进行恢复和清洗, 以便重新使用。分析的地方细胞活动在跑步机揭示了不同的地方领域的机制, 概述了该方法的好处。

一只老鼠被训练在两米长的皮带上运行, 没有提示 (图 1C)。在电极植入后, 一个新的相同长度的带, 但呈现3对线索安装在跑步机上, 为了生成 allocentric 空间表示12,14。宽带信号以3万赫兹的采样率记录, 使用250通道记录系统 (带有 USB 接口板和 custom-made Labview 接口的放大器板) 和两个硅探针 (图 1A, 4 小腿, 每柄8站点) 植入在 CA1 和 CA3 (图 1B)。在高通滤波信号 (0.8-5 赫) 上使用阈值函数检测到单位。使用半自动峰值排序方法15、16、17执行单元隔离。使用 LED 和光传感器的情侣对跑步机的向前/向后运动进行监控, 记录系统的数字输入通道。
每个会话中都可以分离出平均的148±35细胞 (mean±东南亚 m), 其中38.4±3.5% 显示了清晰的位置字段活动 (图 2)。地方领域是相对地稳定横跨许多试验, 在记录的最初的天 (图 2A) 或在几天曝光以后在被暗示的传送带 (图 2B和图 2C)。可以看出不同类型的空间表示。有些单元格显示重复的位置字段与提示的标识 (图 2A) 相关, 而其他单元格显示一个唯一的位置字段 (图 2C)12。因此, 我们可以在数天内记录海马位细胞, 并确定不同的位置场机制, 两个重要的先决条件, 研究与空间导航, 学习和记忆相关的机制和动力学。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56438/56438fig1.jpg"> 
图 1: 硅探针和跑步机设备.(A) 刀柄和硅探针的位置布局。(B) 植入部位。(C) 跑步机设备和皮带上的提示布局。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56438/56438fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56438/56438fig2v2.jpg"> 
图 2: 放置细胞的慢性记录.(a) 在1天记录的单元格示例中, 位置字段 (top)、尖峰 auto-correlogram (左下角) 和尖峰波形 (右下角) 的颜色编码表示。(B-C)与 (A) 3 天 (B) 和5天 (C) 中记录的单元格示例相同。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56438/56438fig2v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Implantation of Chronic Silicon Probes and Recording of Hippocampal Place Cells in an Enriched Treadmill Apparatus at JoVE.com

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

我们描述了植入慢性硅探针的不同步骤, 并记录了老鼠体内的细胞, 这些小鼠在跑步头上固定在一个富含提示的跑步机上。

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 ...

implantation-chronic-silicon-probes-recording-hippocampal-place-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.6K 次观看.  Korea Institute of Science and Technology. 我们描述了植入慢性硅探针的不同步骤, 并记录了老鼠体内的细胞, 这些小鼠在跑步头上固定在一个富含提示的跑步机上。

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

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

科研

神经科学

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

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

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

在麻醉大鼠的硅探头录制的大型神经元合唱

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

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

用于记录单机活动和Electrocorticographic信号的慢性神经电极植入手术

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

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

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<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>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

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 ...

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

光纤植入慢性Optogenetic刺激脑组织

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

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, ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...