Name: recording spatially restricted oscillations in the hippocampus of behaving mice
Uploaded: 2018-07-01T14:30:00.000+00:00
Duration: 7 min 10 s
Description: Watch this Scientific Journal Video about Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice at JoVE.com

我们感谢凯琳温特哈尔特和 Kerstin Semmler 的技术援助。这项工作得到了德国研究基金会 BrainLinks-BrainTools (激动 1086) 的杰出群体的支持。

所有涉及活体动物的方法都已根据《德国动物福利法》获得 Regierungspräsidium 弗赖堡的批准。
1. 筹备工作
<ol><li>设计和建立一个适当的插入工具瞬时携带硅探针和电极连接器在植入过程中。有关自定义内置插入工具的示例, 请参见图 1 。</li>
	<li>小心地释放硅探针和电极连接器从其包装使用陶瓷尖钳。</li>
	<li>提起连接板, 并安全地修复它与一个连接到一个立场的鳄鱼钳。</li>
	<li>使用立体镜, 将探头与插入工具与陶瓷尖钳对准。用一 cauterizer 熔化的2毫米石蜡层将探针粘在插入工具上。在这个过程中注意不要接触探针小腿。</li>
	<li>使用标准胶带将电极连接器固定在插入工具的轴上。请注意, 根据制造商的情况, 接地线可能需要焊接到电极连接板之前植入。用焊料 (400 °c) 的锡焊料, 从两个短的清漆绝缘铜线中去除绝缘。将地线焊接到电极接头板中适当的插槽上。</li>
	<li>除去两块铜线的绝缘。在不锈钢螺丝 (1 毫米直径, 2 毫米长度) 周围环绕每根裸铜线三次。适用于焊接钢的焊剂, 并将铜线焊接到螺钉盖的底部。确保螺纹的下半部分保持无锡焊料。</li>
	<li>使用标准万用表检查电线和螺钉之间的电接触。</li>
	<li>通过浸泡70% 乙醇 (十年代) 对硅探针和地面螺钉的柄进行消毒。</li>
	<li>通过将一个塑料巴斯德吸管的头部切开一半, 为探针植入物准备一个保护罩。</li>
</ol>2. 植入手术
<ol><li>用热珠灭菌器消毒手术器械 (剪刀、细尖钳、手术钳)。用70% 乙醇擦拭所有表面。</li>
	<li>3% 异氟醚在1升/分的氧气中诱导麻醉。<ol>
<li>为维护, 使用 1-1.5% 异氟醚。请注意, 达到手术耐受性所需的异氟醚浓度可以从动物到动物不等。</li>
		<li>当动物无法对脚趾捏起反应时, 就能实现稳定的手术耐受。监测鼠标的呼吸速率, 并在必要时调整异氟醚的浓度。</li>
		<li>在动物的眼睛上涂抹药膏以防止干燥。</li>
	</ol>
</li>
	<li>轻轻地将耳棒插入耳道, 将鼠标装入立体定向的框架中。一旦鼠标的头部由耳棒稳定, 在鼻子上放置一个口片, 连续异氟醚传递。将鼠标放在毛巾上或垫在加热垫上, 并注射丁丙诺啡皮 (0.05-0.1 毫克/千克体重), 以确保术后镇痛。</li>
	<li>用标准剃须刀剃头, 用70% 乙醇消毒皮肤。使用手术剪刀, 在头骨中线切开皮肤, 用手术钳打开皮肤。</li>
	<li>将动物头部与立体定向对齐工具的帮助对准 bregma 和 lambda。在 bregma 和 lambda 之间应该有小于50µm 的高度偏移量。此外, 沿 mediolateral 轴水平测量头部的深度从 bregma 在定义的距离左和右 (例如, 1 毫米左和右 bregma)。如有必要, 调整头部倾斜。</li>
	<li>用3% 过氧化氢清洁头部, 用无菌的棉抹布擦拭乾燥。</li>
	<li>使用适当的立体定向图谱12确定开颅术相对于 bregma 的位置。</li>
	<li>使用0.9 毫米钻头, 在小脑骨上钻两个螺钉孔, 放置地面和参考螺钉。此外, 1-3 孔的锚固螺钉是可取的, 以稳定种植体。定位螺钉的位置将取决于开颅手术的位置。对于植入海马, 放置锚螺钉在对侧壁和同侧额叶皮质。使用合适的螺丝刀将螺钉插入骨骼。注意不要穿透大脑。</li>
	<li>执行开颅手术, 慢慢细化头骨与演习在一个矩形区域周围的植入一侧。经常用灭菌的磷酸盐缓冲剂 (PB) 滋润骨骼。剩下的瘦头骨可以用细 (27G) 注射针和一双镊子轻轻刺穿并取出。</li>
	<li>用薄 (27G) 注射针仔细刺穿硬脑膜。通过用一双镊子弯曲针尖, 并拉硬脑膜去除, 形成一个小钩子。应用 PB 防止大脑表面干燥。</li>
	<li>将电极插入工具安装在一个立体定向的支架上, 将探头移到 bregma 上, 并将探头移动到所述开颅手术的立体定位坐标上。慢慢地穿透大脑表面。请确保探头轴不弯曲。避免通过血管植入。</li>
	<li>慢慢降低探头直到200µm 在所需深度以上。用灭菌的凡士林盖住硅探针的开颅和小腿以保护。应用牙科水泥将探头的底座固定在颅骨的锚固螺钉上。</li>
	<li>在水泥应用之后, 慢慢地将探头移至目标深度。在水泥应用后推进最后的200µm, 减少了探针的侧向运动, 确保了靶区组织损伤最小。请注意, 使用的水泥固化时间会影响协议的这一步骤。用快速固化水泥, 省略此步骤, 直接将探头植入目标深度, 以避免对硅探针的损坏。</li>
	<li>水泥固化后, 用 cauterizer 将蜡熔化, 从插入工具中释放探针。</li>
	<li>将连接器板从插入设备上松开, 并将其放置在头骨的适当位置, 使用附着在插入手柄上的鳄鱼钳。如果探头植入海马, 将连接器板放在侧壁骨上。用牙科水泥将连接板固定在头骨上。</li>
	<li>将连接器板的地线和参考线焊接到连接在小脑上的两个螺钉上的导线上。</li>
	<li>将防护罩修剪到正确的高度, 并将其置于硅探针上。用牙科水泥把盖子固定在连接板和头骨上, 避免裸露头骨周围的皮肤。通常不需要在植入部位周围缝合皮肤。</li>
</ol>3. 手术后的恢复
<ol><li>应用适当的镇痛治疗至少2天 (如皮下注射丁丙诺啡每6小时在白天和在饮用水中过夜结合卡洛芬 (4-5 毫克/千克体重) 每 24 h)。建议单壳防止对植入物造成损害。</li>
	<li>允许至少一周的恢复。与当地动物福利指南进行协商。</li>
</ol>4. 数据采集
<ol><li>记录 LFPs 从自由移动的小鼠使用一个适当的数据采集系统通过换向器连接。要获取 LFPs, 请使用 1-5 赫的采样频率。如果单单元放电与 LFP 一起记录, 则需要较高的采样率 (20-30 赫)。</li>
	<li>存储单个通道的原始记录文件以进行离线分析。</li>
</ol>5. 组织学
<ol><li>录音完成后, 深麻醉动物 (如2 克/千克体重聚氨酯注射腹腔)。通过对脚趾捏缺乏反应来确认麻醉状态。</li>
	<li>灌注老鼠 transcardially 与冰冷磷酸盐缓冲盐水 (~ 1 分钟) 跟随4% 多聚甲醛 (~ 10 分钟) 使用标准心内灌注方法13。在灌注前, 记录站点的电解毁损可能通过执行 (例如, 通过应用 10-20 V 的恒定电压, 最多1秒)。另外, 在植入前, 使用的荧光染料可以用来追踪识别。建议采用不同类型的硅探针检测电极位置的各种识别方法, 以获得最佳结果。</li>
	<li>切脑部分 (~ 100 µm) 和染色切片与 4 '-6-diamidino 2-苯基吲哚 (DAPI, 1 µg/毫升) 后, 三洗涤步骤 PB (每10分钟室温)。</li>
	<li>将切片放在显微镜幻灯片上, 应用嵌入介质的一滴, 并用盖子滑动覆盖截面。让嵌入介质在室温下隔夜干燥。</li>
	<li>使用荧光或共焦激光扫描显微镜, 确定记录站点的位置。</li>
	<li>要想进一步使用硅探针, 请用一个鳄鱼钳握住探针, 然后用烙铁 (400 °c) 小心地将牙科水泥熔化, 从而释放探针。在这个过程中注意不要接触探针柄!</li>
	<li>在热蒸馏水 (80 °c, 15 分钟) 内冲洗探针, 然后用酶溶液 (1% Tergazyme 在蒸馏水中, 室温30分钟) 和蒸馏水中的另一洗涤步骤 (15 分钟)。请注意, 探针恢复的成功率很低。</li>
</ol>6. 惩教署分析
<ol><li>使用适当的分析环境 (如Python), 将单个小腿的 LFP 数据转换为 CSD, 方法是将小腿上的第二个空间导数近似为 <img alt="Equation" src="/files/ftp_upload/57714/57714eq1.jpg"> 当LFPn, t 是n次电极上的 LFP 信号时t和Δz是电极间间距。请注意, 由于n-1 和n+1 操作, 不能估计小腿的第一和最后电极的 CSD, 在探针安置期间必须考虑到。使用代码的短段来实现近似公式, 在迭代期间计算每个电极的 CSD 信号 (请参见补充代码文件)。</li>
	<li>使用获得的 CSD 信号进行进一步分析 (例如,通过应用带通滤波器研究脑振荡的特定频段)。</li>
</ol>

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作者没有什么可透露的。

增加的证据表明, 海马神经元电路的脑振荡发生在离散空间域10,11,16。CSD 分析大大降低了容积传导的影响, 是研究局部振荡事件的重要前提。通过这个视频, 我们提供了一个指南, 以植入小鼠海马的硅探针, 以分析惩教署的数据。我们展示了 CA1 中尖锐波波纹和齿状回局部伽玛振荡信号的代表性例子。然而, 这个协议也可以用来研究其他海马振荡活动模式, 如θ或与呼吸相关的网络振荡17。
植入的成功关键在于在立体定向的框架中正确地对准动物的头部。我们使用注射针夹在一个立体定向的持有人测量的偏差, 头部在 anterioposterior 和 mediolateral 方向。我们按顺序移动针以接触 bregma 和 lambda, 并补偿两点高度之间的任何偏移通过倾斜的立体定向的框架。同样, 测量的高度在1毫米左右的 bregma 将表明任何 mediolateral 偏移, 这可以通过倾斜的框架左或右调整。补偿 < 50 µm 推荐最佳植入效果。
探头设计的选择是另一个关键的方面。我们的模拟结果表明, 分离局部事件的能力随着电极间距的增加而下降。我们成功地隔离了局部伽玛振荡事件使用硅探针与25和100µm 电极间距和250和400µm 小腿间距11。这些指标为探针设计提供了一个良好的出发点。
鉴于硅探针的高成本, 记录探头的可用性目前是一个限制因素。此处描述的方法允许主探测器恢复。但是, 我们只在一个实例中成功地恢复了探测器的记录, 表明该程序的成功率很低。今后对该议定书的改进可能包括使用旨在促进探测器恢复18的微型驱动器。

LFP 中的振荡在神经回路的信息处理中有重要的介入。它们涵盖了广泛的频率范围, 从慢波 (1 赫兹) 到快速脉动振荡 (~ 200 赫兹)1。不同频段与认知功能相关, 包括记忆、情感处理和导航2、3、4、5、6、7。电流在神经元膜上的流动构成了 LFP 信号8的最大部分。阳离子进入细胞 (例如通过激活谷氨酸兴奋突触) 代表一个活跃的电流接收器 (电荷离开胞外培养基)。相比之下, 正电荷对胞外介质的净流量, 例如通过激活 GABAergic 抑制突触, 描述了该位置的活动电流源。在神经元偶极子中, 电流汇与被动源成对, 反之亦然, 因为补偿电流影响到远处的膜电荷。
由远程神经过程产生的电场也可能导致记录电极上的电压偏转相当大, 因此可能被错误地认为是局部事件。这种体积传导对 LFP 信号的解释构成了严重的挑战。惩教署的分析提供有关本地电流汇和 LFP 信号来源的资料, 因此包括减少体积传导8的影响的方法。在层状结构, 如海马体, 一维的 CSD 信号可以获得的第二空间导数的 LFP 记录从等距电极安排垂直于层流平面9。商用线性硅探针的问世使研究人员得以利用 CSD 方法研究海马区的局部振荡活动。例如, 已经证明, 不同的伽玛振荡在 CA1 区10中以特定层的方式出现。此外, 惩教署的分析已确定的独立热点的伽玛活动在主细胞层的齿状回11。重要的是, 这些发现只在当地的惩教署, 而不是 LFP 的信号中可见。因此, 惩教署的分析提供了一个强有力的工具, 以获得洞察的电路操作海马。
在本协议中, 我们提供了一个全面的指南, 以获得一维的 CSD 信号与硅探针。这些方法将使用户能够调查行为小鼠海马区内的局部振荡事件。

<table><tbody><tr><td>Crocodile clamp with stand</td><td>Reichelt Elektronik</td><td>HALTER ZD-10D</td><td></td></tr><tr><td>Silicon probe</td><td>Cambridge Neurotech</td><td>P-series 32</td><td></td></tr><tr><td>Stereoscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Varnish-insulated copper wire</td><td>B&amp;uuml;rklin Elektronik</td><td>89 F 232</td><td></td></tr><tr><td>Ground screws</td><td>Screws &amp;amp; More GmbH (screwsandmore.de)</td><td>DIN 84 A2 M1x2</td><td></td></tr><tr><td>Flux</td><td>Stannol</td><td>114018</td><td></td></tr><tr><td>Ceramic-tipped forceps</td><td>Fine Science Tools</td><td>11210-60</td><td></td></tr><tr><td>Paraffine Wax</td><td>Sigma-Aldrich</td><td>327204</td><td></td></tr><tr><td>Cauterizer</td><td>Fine Science Tools</td><td>18010-00</td><td></td></tr><tr><td>Soldering iron</td><td>Kurtz Ersa</td><td>OIC1300</td><td></td></tr><tr><td>Multimeter</td><td>Uni-T</td><td>UT61C</td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth</td><td>9065.1</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Carl Roth</td><td>EA65.1</td><td></td></tr><tr><td>Heat sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Stereotaxic frame</td><td>David Kopf</td><td>Model 1900</td><td></td></tr><tr><td>Stereotaxic electrode holder</td><td>David Kopf</td><td>Model 1900</td><td></td></tr><tr><td>Isoflurane</td><td>Abbvie</td><td>B506</td><td></td></tr><tr><td>Oxygen concentrator</td><td>Respironix</td><td>1020007</td><td></td></tr><tr><td>Buprenorphine</td><td>Indivior UK Limited</td><td></td><td></td></tr><tr><td>Electrical shaver</td><td>Tondeo</td><td>Eco-XS</td><td></td></tr><tr><td>Heating pad</td><td>Thermolux</td><td>463265/-67</td><td></td></tr><tr><td>Surgical clamps</td><td>Fine Science Tools</td><td>18050-28</td><td></td></tr><tr><td>Hydrogen peroxide</td><td>Sigma-Aldrich</td><td>H1009</td><td></td></tr><tr><td>Sterile cotton wipes</td><td>Carl Roth</td><td>EH12.1</td><td></td></tr><tr><td>Drill</td><td>Proxxon</td><td>Micromot 230/E</td><td></td></tr><tr><td>21G injection needle</td><td>B. Braun</td><td>4657527</td><td></td></tr><tr><td>Phosphate buffer/phosphate buffered saline</td><td></td><td></td><td></td></tr><tr><td>Stereotaxic atlas</td><td>Elsevier</td><td>9.78012E+12</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14094-11</td><td></td></tr><tr><td>Surgical forceps</td><td>Fine Science Tools</td><td>11272-40</td><td></td></tr><tr><td>27G injection needles</td><td>B. Braun</td><td>4657705</td><td></td></tr><tr><td>Vaseline</td><td></td><td></td><td></td></tr><tr><td>Dental cement</td><td>Sun Medical</td><td>SuperBond T&amp;amp;M</td><td></td></tr><tr><td>Carprofen</td><td>Zoetis</td><td>Rimadyl 50mg/ml</td><td></td></tr><tr><td>Recording amplifier</td><td>Intan Technologies</td><td>C3323</td><td></td></tr><tr><td>USB acquisition board</td><td>Intan Technologies</td><td>C3004</td><td></td></tr><tr><td>Recording cables</td><td>Intan Technologies</td><td>C3216</td><td></td></tr><tr><td>Electrical commutator</td><td>Doric lenses</td><td>HRJ-OE_FC_12_HARW</td><td></td></tr><tr><td>Acquisition software</td><td>OpenEphys (www.open-ephys.org)</td><td>GUI</td><td>allows platform-independent data acquisition</td></tr><tr><td>Computer for data acquisition</td><td></td><td></td><td></td></tr><tr><td>Analysis environment</td><td>Python (www.python.org)</td><td></td><td>allows platform-independent data analysis</td></tr><tr><td>Urethane</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000</td><td></td></tr><tr><td>Microscope slides</td><td>Carl Roth</td><td>H868.1</td><td></td></tr><tr><td>Cover slips</td><td>Carl Roth</td><td>H878.2</td><td></td></tr><tr><td>Embedding medium</td><td>Sigma-Aldrich</td><td>81381-50G</td><td></td></tr><tr><td>Distilled water</td><td>Millipore</td><td>Milli Q</td><td>Table-top machine for the production of distilled water</td></tr><tr><td>Tergazyme</td><td>Alconox</td><td>Tergazyme</td><td></td></tr></tbody></table>

recording spatially restricted oscillations in the hippocampus of behaving mice

局部场电位 (LFP) 是通过神经膜的离子运动而产生的。由于 LFP 电极所记录的电压反映了大量脑组织的电场, 提取有关当地活动的信息具有挑战性。然而, 研究神经元集成电路需要对真正的局部事件和来自遥远脑区的体积传导信号进行可靠的区分。电流源密度 (CSD) 分析通过提供有关电极附近的电流汇和源的信息, 为这个问题提供了解决方案。在具有层流细胞构筑的脑区, 如海马, 可通过估计 LFP 的第二空间导数来获得一维 CSD。在这里, 我们描述了一个方法, 记录 multilaminar LFPs 使用线性硅探针植入到背海马。惩教署的踪迹沿探头的个别柄计算。该协议描述了一种解决自由移动小鼠海马空间受限神经元网络振荡的方法。

图 1说明了插入工具用于硅探针的植入。图 2显示了长期植入的硅探针靶向 CA1 区和齿状回颗粒细胞层的记录。我们在 homecage 的自由运动过程中记录了探针柄 LFPs。为了最小化体积传导的影响, 所得到的信号被转换为 CSD 沿探针的每个柄 (图 2B, D)。在图 2B所示的第一个例子中, 单一的尖锐波波纹事件导致了 CA1 地层 radiatum 中的一个突出的电流下沉。在图 2C中显示的第二个例子中, 我们记录了从齿状回 (侧距400µm) 颗粒细胞层中的两个不同的部位。高伽玛波段通过对 CSD 信号应用 60-80 Hz 带通滤波器隔离, 在颗粒细胞层中的两个记录点之一上暴露出局部伽玛爆发 (图 2D; 红色记录站点)。请注意, 在将记录信号转换为 CSD 后, 只能检测到局部伽玛振荡活动 (图 2D, 右)。图 2E显示同一记录的不同时间段, 在此期间, 两个记录站点上的伽玛振荡在其相位 (图 2E、左) 或振幅 (图 2E、中间) 中不同。最右边的示例显示两个记录站点上同步伽玛活动的纪元。这些例子说明, 惩教署的分析, 不仅可以在分析的维度, 而且在垂直方向上孤立局部活动。为了进一步说明这个中心概念, 我们用一个由两个柄 (I 和 II) 组成的硅探针模拟了一个实验, 每个五个记录点都与图 2所示的结果类比。在我们的模型中, 探针被植入到齿状回中, 局部伽玛振荡出现在颗粒细胞层旁, 记录4的小腿 I (图 3)。假设均匀的体积传导整个组织, 局部振荡将记录在 LFP 在所有其他记录网站的振幅过滤版本 (图 3C)。然而, 沿两个小腿进行的 CSD 分析清楚地隔离了伽玛世代的轨迹 (图 3D)。此外, 同一柄上记录站点之间的距离越小, 在相邻小腿上的 CSD 信号之间的串扰就越小 (图 3E、F)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57714/57714fig1.jpg"> 图 1: 插入工具的图像.硅探针附着在一根粘在鳄鱼钳底座上的昆虫别针上。该支架的轴可以插入一个立体定向的微型机械手植入。<a href="https://www.jove.com/files/ftp_upload/57714/57714fig1large.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/57714/57714fig2.jpg"> 图 2: 惩教署在海马区分析的代表性结果.a: 由四柄硅探针 (柄间距400µm, 电极间距100µm, 8 电极/小腿) 组成的电极阵列被植入到区域 ammonis 1 (CA1)。小腿 A-C 导线地层 oriens (o), 地层 pyramidale (p) 和地层 radiatum (r)。HF: 海马裂。有齿状回。B: LFP (黑色痕迹) 和 CSD (彩色编码) 的三小腿在 CA1 的200毫秒的时代, 其中包含自发的尖锐波波纹事件。注意地层 radiatum 中的突出电流下沉。LFP 刻度条: 2 mV。C: 来自 DG 的硅探针记录的示例。小腿 A 和 B 穿透 DG。CA3: ammonis 3 区。电解损伤后进行记录, 以标签电极轨道。D: 电极小腿的图画与海马解剖学有关。LFP 在颗粒细胞层 (红色和蓝色记录站点) 上从两个空间上分离的站点记录, 表明伽玛振荡 (60-80 Hz) 在两个站点之间 (左) 同步。然而, 在同一时间间隔的 CSD 信号显示, 一个焦点伽玛 ' 热点 ' 限制在红色记录站点 (右)。惩教署在两个录音地点发出的讯号均以相同的比例绘制。E: CSD 但不 LFP 信号识别两个记录站点之间的相位 (左) 和振幅异步 (中间) 段。右边的纪元显示了同步伽玛活动的一个短暂的时代。底部的痕迹说明相差 (Δ相位以弧度表示) 和振幅差分指数 (一种现代指数), 定义为两个记录点之间的振幅差除以每一个时间点的振幅和从-1 到1的幅度的总和)LFP (灰色) 和 CSD (黑色) 痕迹。所有三个例子都是30毫秒长, 并发生在400毫秒内. C 和 D 在创意共同性 (https://creativecommons.org/Licenses/by/4.0/) 下由 Strüber等201711改编。两个录音地点的惩教署讯号均以同一比例在每个个别小组抽取。<a href="https://www.jove.com/files/ftp_upload/57714/57714fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57714/57714fig3.jpg"> 图 3: 在齿状回颗粒细胞层中模拟焦40赫兹振荡的模型实验。a: 在 d柄的距离上有两柄的模型硅探头 = 400 µm 和五个记录点, 位于 d电极的距离 = 100 µm 放置在齿状回。B: 在记录4的小腿 I 的位置, 一个焦40赫兹振荡被模拟为正弦波形加上高斯白噪声。在所有其他网站上, 只会引起噪音。参数: 伽玛振幅: 1 mV;噪音标准差: 0.05 mV;世俗决议:10 赫。C: 我们用简化的方法模拟了体积传导通过细胞外空间的振幅滤波效应, 作为伽玛振幅的负指数衰减, 其组织空间常数为500µm。正确描述 LFP 信号的横向扩散的确切参数是高度争议的14。然而, 我们的估计很适合从皮层伽玛振荡相干性研究15的数据。在产生的 LFP 中, 最初的震源振荡包括几个记录点。D: 在使用给定的 csd 方程对单个轴执行 csd 分析后, 振荡仅在原始位置可见。E:LFP (左、红) 和 CSD 的功率谱密度分析 (右, 蓝) 记录站点4在小腿 I (虚线) 和 II (连续线)。请注意, CSD 分析正确隔离了小腿 I 上的局部40赫兹振荡, 而 LFP 信号在小腿 ii 上包含大量的音量传导振荡活动。采用 MATLAB 的 pwelch 函数进行 PSD 分析。F:将单个小腿上的录音点之间的距离增加到400µm, 从而降低了 CSD 分析分离病灶活动的能力。采用 MATLAB 7.10 进行了仿真和分析。<a href="https://www.jove.com/files/ftp_upload/57714/57714fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice at JoVE.com

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

该协议描述了用多柄线性硅探针记录局部场电位的情况。利用电流源密度分析转换信号, 可以重建小鼠海马区的局部电活动。利用这种技术, 可以在自由运动小鼠中研究空间限制的脑振荡。

行为小鼠海马空间受限振荡的记录

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed ...

recording-spatially-restricted-oscillations-hippocampus-behaving

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.8K 次观看.  University of Freiburg. 该协议描述了用多柄线性硅探针记录局部场电位的情况。利用电流源密度分析转换信号, 可以重建小鼠海马区的局部电活动。利用这种技术, 可以在自由运动小鼠中研究空间限制的脑振荡。

视频: Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

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 ...

科研

神经科学

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 在行为小鼠海马θ振荡中的作用

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

急性小鼠脑切片研究自发海马网络活动

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

行为小鼠希波坎帕的神经元活动可视化的颅骨切除术

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

小鼠大脑中的 LFP 记录用于认知研究和药物分析

Dual Extracellular Recordings in the Mouse Hippocampus and Prefrontal Cortex

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal populations. It serves as a crucial tool in cognitive research, particularly in brain regions like the hippocampus and prefrontal cortex. Dual LFP recordings between these areas are of particular interest as they allow the exploration of interregional signal communication. However, methods for performing these recordings are rarely described, and most commercial recording devices are either expensive or lack adaptability to accommodate specific experimental designs. This study presents a comprehensive protocol for performing dual-electrode LFP recordings in the mouse hippocampus and the prefrontal cortex to investigate the effects of antipsychotic drugs and potassium channel modulators on LFP properties in these areas. The technique enables the measurement of LFP properties, including power spectra within each brain region and coherence between the two. Additionally, a low-cost, custom-designed recording device has been developed for these experiments. In summary, this protocol provides a means to record signals with high signal-to-noise ratios in different brain regions, facilitating the investigation of interregional information communication within the brain.

作者聚焦:使用双 LFP 记录方案研究药物对小鼠大脑信号的影响

This protocol outlines the use of a custom-designed recording device and electrodes to record local field potentials and investigate information flow in the mouse hippocampus and prefrontal cortex.

小鼠海马体和前额叶皮层中的双重细胞外记录

The technique of recording local field potentials (LFPs) is an electrophysiological method used to measure the electrical activity of localized neuronal ...

<meta charset="utf8"></head><body>使用 Miniscope 成像记录气味羽流导航过程中的神经元活动

Miniscope Recording Calcium Signals at Hippocampus of Mice Navigating an Odor Plume

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to monitor behavior and record Ca2+ transients in dorsal CA1 stratum pyramidale neurons in the hippocampus (dCA1) in mice navigating an odor plume in a 50 cm x 50 cm x 25 cm odor arena. An epifluorescence miniscope focused through a gradient-index (GRIN) lens imaged Ca2+ transients in dCA1 neurons expressing the calcium sensor GCaMP6f in Thy1-GCaMP6f mice. The paper describes the behavioral protocol to train the mice to perform this odor plume navigation task in an automated odor arena. The methods include a step-by-step procedure for the surgery for GRIN lens implantation and baseplate placement for imaging GCaMP6f in CA1. The article provides information on real-time tracking of the mouse position to automate the start of the trials and delivery of a water reward. In addition, the protocol includes information on using an interface board to synchronize metadata describing the automation of the odor navigation task and frame times for the miniscope and a digital camera tracking mouse position. Moreover, the methods delineate the pipeline used to process GCaMP6f fluorescence movies by motion correction using the NorMCorre algorithm followed by identification of regions of interest with EXTRACT. Finally, the paper describes an artificial neural network approach to decode spatial paths from CA1 neural ensemble activity to predict mouse navigation of the odor plume.

<meta charset="utf8"></head><body>作者聚焦:通过实时行为分析了解大脑对嗅觉和空间信息的处理

This protocol investigates the brain-behavior relationship in hippocampal CA1 in mice navigating an odor plume. We provide a step-by-step protocol, including surgery to access imaging of the hippocampus, behavioral training, miniscope GCaMP6f recording and processing of the brain, and behavioral data to decode the mouse position from ROI neural activity.

微型显微镜记录在气味羽流中导航的小鼠海马体的钙信号

Mice navigate an odor plume with a complex spatiotemporal structure in the dark to find the source of odorants. This article describes a protocol to ...

行为小鼠海马空间受限振荡的记录

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小鼠海马体和前额叶皮层中的双重细胞外记录

小鼠海马体和前额叶皮层中的双重细胞外记录

小鼠海马体和前额叶皮层中的双重细胞外记录

微型显微镜记录在气味羽流中导航的小鼠海马体的钙信号

微型显微镜记录在气味羽流中导航的小鼠海马体的钙信号