这项工作得到了国家自然科学基金（31871170、32170950和31970915）、广东省自然科学基金（2021A1515010804和2023A1515010899）、广东省自然科学基金重大培育项目（2018B030336001）和广东省资助:脑疾病治疗关键技术（2018B030332001）的资助。

所有小鼠均在商业上获得，并在室温为22-25°C和相对湿度为50%-60%的情况下，在12小时光照/ 12小时黑暗循环（当地时间上午08:00点亮）中维持。小鼠可以获得持续的食物和水供应。所有实验均按照华南师范大学《实验动物护理与使用指南》进行，并经机构动物伦理委员会批准。以3-5月龄雄性C57BL/6J小鼠为研究对象。
1.微型驱动系统总成
<ol><li>使用两个支架和一个固定可移动微型驱动器的螺钉连接两块计算机设计的板，并将连接器连接到一块板上（图 1A，Bi-iii）。通过拧动螺钉（0.5 毫米/圈）驱动微型驱动器。</li>
	<li>确保微型驱动器可以携带两组八根导管（~3 cm 长，~50 μm 内径，~125 μm 外径）用于 MC 区域的每一侧，然后将其切割成相同的长度（至少 15 mm;图1Biv，v）。</li>
	<li>切割 16 根直径为 35 μm 的镍铬线（~5 cm 长），然后将它们依次装入导管中，然后涂上胶水固定它们（图 1Bi、vi、vii）。</li>
	<li>剥去电线绝缘层，按照通道图将每根裸露的电线依次缠绕到连接器的每个引脚上，以及参考电极和接地电极，然后慢慢地在每个引脚上涂上导电漆（图 1Bviii-x）。</li>
	<li>使用环氧树脂覆盖引脚（图1Axi，xii），然后通过阻抗测试仪进行镀金，将电极尖端的阻抗降低到~350 kOhm（图1Bxiii，xiv）。设置阻抗测试仪的参数如下:-10.08 μA 直流电，持续 1 秒，镀金溶液，包括 5 mM PtCl4。</li>
	<li>最后，拧动螺丝将微型驱动器移至顶部。检查 如图 1A 所示修改的微型驱动系统的整体尺寸（约 15 mm 长、10 mm 宽、20 mm 高、~1 g 重量）。检查 图 1Ai、ii 中计算机设计的电路板和可移动组件的详细规格。</li>
</ol>2. 电极阵列植入
<ol><li>在手术开始前对手术包进行消毒，戴上无菌手套并穿上医生的无菌白大褂。</li>
	<li>为了控制疼痛，在诱导室中为小鼠使用皮下注射美洛昔康注射剂（5mg / kg）。然后通过在诱导室18,19中腹膜内（ip）注射戊巴比妥（80mg / kg）麻醉小鼠。如果脚趾捏反射仍然存在，则应用补充剂量的戊巴比妥 （20 mg/kg/h）。</li>
	<li>将鼠标固定在立体定位装置中，并使用温度控制器将其直肠温度保持在37°C。</li>
	<li>将四环素眼膏涂抹在小鼠的双眼上，并在手术前再次更换无菌手套。</li>
	<li>剃掉小鼠的皮毛，并使用无菌棉签涂抹器以同心圆向外移动，用三轮交替的betadine磨砂膏和酒精对手术部位进行消毒（图2i，ii）。做一个小的中线切口（~15 毫米）以暴露其头骨。立即将 1% 利多卡因局部涂抹在颈部肌肉上以缓解疼痛。然后，用剪刀去除残留的组织，并使用涂有盐水的棉签清洁头骨（图2iii）。</li>
	<li>使用充满墨水的玻璃微电极，标记双侧MC的所需位置以进行植入（图2iv，v）。根据先前的研究20，双侧 MC 的位置如下:前囟前方 0.74 mm，中线外侧 1.25 mm。</li>
	<li>植入四个不锈钢螺钉（直径0.8毫米）以保护微驱动系统，然后将所有螺钉与参比电极和接地电极连接在一起，然后用混合牙科水泥覆盖以形成壁（图2vi-习）。</li>
	<li>在MC区域的协调头骨的左右两侧用颅骨钻仔细钻两个小孔（~1.5mm2）（图2xii）。使用双侧 MC 的立体定位坐标:前囟前方 0.74 mm，中线外侧 1.25 mm，硬脑膜腹侧 0.5 mm。</li>
	<li>用细镊子小心地从孔中取出硬脑膜（图2xiii）。</li>
	<li>使用显微操纵器以 10 μm/s 的速度将微驱动系统插入孔的中心（图 2xiv-xvii）。</li>
	<li>完成微型驱动系统的插入后，将凡士林填充到牙科水泥壁中（图2xviii）。</li>
	<li>将微型驱动系统的底板和牙科水泥壁与混合牙科水泥连接起来（图2xix）</li>
	<li>用生理盐水清洗切口，然后用含有盐酸林可霉素和盐酸利多卡因的凝胶进行局部治疗，以缓解术后疼痛。</li>
	<li>将导电铜箔带缠绕在植入的微型驱动系统周围（图 2xx）。</li>
	<li>将鼠标移入保持在31-33°C的笼子中，并监测鼠标从麻醉中恢复。</li>
	<li>通过单独喂食让小鼠恢复1周。检查并用含有盐酸林可霉素和盐酸利多卡因的凝胶连续应用3天来治疗切口。</li>
</ol>3. 自由移动小鼠双侧 MC 的多通道记录
<ol><li>轻轻小心地握住清醒的鼠标的头部。至少提前 0.1 天扭转微型驱动系统可移动部分上的螺钉（图 1Aii），向下移动电极阵列（~1 mm 深度）。</li>
	<li>轻轻小心地握住清醒鼠标的头部。用螺纹连接头级的中心和氦气球（充满约 0.02 L 氦气），以抵消头级和微型驱动系统的重量（图 3A、B）。</li>
	<li>通过在记录软件中以 30 kHz 采样，使用记录电极和多通道系统捕获原始信号，然后使用多通道系统中的数模 （DA） 转换器进行数字化。</li>
	<li>通过在记录软件中以 10 kHz 重新采样，从原始数据中提取 LFP 信号，然后使用记录软件中的陷波滤波器去除 50 Hz 线路噪声。</li>
	<li>从自由移动的鼠标中以稳定状态记录原始数据至少 60 秒。完成录制后，慢慢断开头戴式和微型驱动系统之间的连接，并将鼠标放回原位。</li>
	<li>将记录的数据存储在计算机中并离线分析（图4 和 图5）。</li>
	<li>完成实验后，按照研究所的指南进行安乐死，然后使用3V输出的电源确认电极的位置，进行1分钟的电解病变，然后进行组织学分析。使用冷冻切片机将小鼠的大脑切成30μm切片，收集MC切片，然后用显微镜捕获图像（图3C，D）。</li>
</ol>4. 尖峰分选和分析
<ol><li>在尖峰排序软件 中单击"文件">"打开> Nev文件 "，打开以30 kHz采样的尖峰数据（图4Ai）。</li>
	<li>单击"信息"以选择未排序的通道，然后选择 "排序">"更改排序方法">"使用 K-Means"。按下按钮 Valley-Seeking Sort > K-Means Sorting 以获得排序单位（图 4Aii、iii）。</li>
	<li>单击 "文件">"另存为"，以.nev文件扩展名保存排序后的尖峰数据，然后选择 "文件">"导出每个波形数据 "，以.txt文件扩展名导出PCA结果（图4Aiv）。</li>
	<li>单击 "文件">"将数据导入>Blackrock文件 "，进行神经生理学数据分析，打开排序后的尖峰文件（图4Bi）。</li>
	<li>单击"分析>自相关图"以获取所选单元的自相关图，然后按如下方式设置参数:−0.05 s处的X最小值，0.05 s处的X最大值和0.001处的Bin值（图4Bii，iii）。</li>
	<li>加载排序后的尖峰数据，单击 "分析>尖峰间间隔直方图 "以获取尖峰间间隔直方图，然后设置如下参数:0 秒处的最小间隔值、0.1 秒处的最大间隔值和 0.001 处的 Bin 值（图 4Biv、v）。</li>
	<li>单击" 分析>交叉相关图 "以获取两个排序单元事件之间的交叉相关图，然后按如下方式设置参考事件和参数:−0.1 s 处的 X 最小值、0.1 s 处的 X 最大值和 0.001 处的 Bin 值（图 4Bvi、vii）。</li>
	<li>单击 "结果">"数值结果 "以保存具有.xls文件扩展名的自相关图、尖峰间间隔直方图和交叉相关图的结果（图 4Bviii、ix）。分析数据，并绘制图表。</li>
</ol>5. LFP分析
<ol><li>在软件中单击 文件导入数据>Blackrock文件 进行神经生理学数据分析，以打开以10 kHz采样的连续信号数据（图5Ai）。</li>
	<li>单击 Analysis > Spectrum for Continuous 以分析所选通道的 LFP 功率谱。按如下方式设置参数:频率值数为 8,192，多重锥度值为 3-5，归一化百分比占总功率谱密度 （PSD） 的百分比，频率范围为 1 Hz 至 100 Hz（图 5Aii、iii）。</li>
	<li>单击" 连续>相干分析"（Analysis Coherence for Continuous）， 分析 MC 左侧和右侧两个 LFP 的相干性。 按如下方式设置参考通道和参数: 在相干值、频率值数 8,192、多个锥度值在 3-5 和频率范围 1 Hz 至 100 Hz 处计算（图 5Aiv， v）。</li>
	<li>单击" Analysis > Corr. with Cont. Variables "，分析 MC 左右两侧两个 LFP 之间的相关性。 设置参考通道（LFP 数据）和参数如下:−0.1 s 处的 X 最小值、0.1 s 处的 X 最大值和 0.001 处的 Bin 值（图 5Avi， vii）。</li>
	<li>单击"结果">"数值结果"，将PSD、一致性和相关性的结果与.xls文件扩展名一起保存（图5Aviii，ix）。</li>
	<li>选择每个频段需要提取代表性迹线的信道，点击"连续变量数字滤波编辑">得到每个频带，然后设置参数如下:滤波器频率响应为带通，滤波器实现为无限脉冲响应（IIR）巴特沃斯，滤波器阶数值为2。最后，设置感兴趣的频率范围（图5Bi-iv）。 注意:此处使用的频率范围如下:δ（δ，1-4 Hz）、θ（θ，5-12 Hz）、β（β，13-30 Hz）、低伽马（低γ，30-70 Hz）和高伽马（高γ，70-100 Hz）振荡。</li>
	<li>分析数据并绘制图表。</li>
</ol>6. 尖峰与LFP之间的相关性
<ol><li>在软件中点击 "文件">"导入数据>贝莱德文件 "进行神经生理学数据分析，打开连续信号数据和尖峰数据。</li>
	<li>单击 "分析">"相干分析 "以分析所选通道的尖峰和 LFP 之间的相干性。设置参考变量（尖峰时序）和参数，如下所示:在相干值、频率值数 512、多重锥度值 3-5 以及频率范围为 1 Hz 至 100 Hz 处计算（图 5Ci、ii）。</li>
	<li>单击"结果">"数值结果"，以.xls文件扩展名保存尖峰场相干性的结果（图5Ciii，iv）。</li>
	<li>分析数据并绘制图表。</li>
</ol>

小鼠运动皮层中的细胞外电生理记录

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A., Gail, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+open-source+toolboxes+for+processing+and+analysis+of+spike+and+local+field+potentials+data.">Comparing open-source toolboxes for processing and analysis of spike and local field potentials data.</a> Frontiers in Neuroinformatics. 13, 57 (2019).</li><li>Mao, L., Wang, H., Qiao, L., Wang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Nrf2+enhances+the+upregulation+of+nuclear+factor-kappaB+activity,+tumor+necrosis+factor-alpha,+and+matrix+metalloproteinase-9+after+spinal+cord+injury+in+mice.">Disruption of Nrf2 enhances the upregulation of nuclear factor-kappaB activity, tumor necrosis factor-alpha, and matrix metalloproteinase-9 after spinal cord injury in mice.</a> Mediators of Inflammation. 2010, 238321 (2010).</li><li>Jin, Z., Zhang, Z., Ke, J., Wang, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-linked+irisin+prevents+mortality+and+enhances+cognition+in+a+mice+model+of+cerebral+ischemia+by+regulating+Klotho+expression.">Exercise-linked irisin prevents mortality and enhances cognition in a mice model of cerebral ischemia by regulating Klotho expression.</a> Oxidative Medicine and Cellular Longevity. 2021, 1697070 (2021).</li><li>Ding, X., 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=Spreading+of+TDP-43+pathology+via+pyramidal+tract+induces+ALS-like+phenotypes+in+TDP-43+transgenic+mice.">Spreading of TDP-43 pathology via pyramidal tract induces ALS-like phenotypes in TDP-43 transgenic mice.</a> Acta Neuropathologica Communications. 9 (1), 15 (2021).</li><li>Cao, W., 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=Gamma+oscillation+dysfunction+in+mPFC+leads+to+social+deficits+in+neuroligin+3+R451C+knockin+mice.">Gamma oscillation dysfunction in mPFC leads to social deficits in neuroligin 3 R451C knockin mice.</a> Neuron. 97 (6), 1253-1260 (2018).</li></ol>

作者没有什么可透露的。

在自由移动的小鼠中，多通道记录被认为是神经科学研究中的一项有用技术，但对于初学者来说，记录和分析信号仍然相当具有挑战性。在本研究中，我们提供了制造微驱动系统和进行电极植入的简化指南，以及 通过 尖峰分选软件和神经生理学数据分析软件捕获和分析电信号的简化程序。
鉴于定制微驱动系统的质量极大地有助于在自由移动的小鼠 14、15<...

局部场电位 （LFP） 是细胞外信号的重要组成部分，反映了大量神经元的突触活动，这些神经元构成了多种行为的神经代码1。神经元活动产生的尖峰被认为有助于 LFP，并且对神经编码很重要2.棘峰和 LFP 的改变已被证明可以介导多种脑部疾病，例如阿尔茨海默病，以及恐惧等情绪3,4。值得注意的是，许多研究都强调，动物清醒和麻醉状态之间的尖峰活性显着不同5.尽管麻醉动物的记录提供了一个机会，可以在高度定义的皮质同步状态下以最小的伪影评估 LFP，但结果在一定程度上与清醒受试者 6,7,8 中的结果不同。因此，使用植入大脑的电极在清醒的大脑状态下检测各种疾病在长时间尺度和大空间尺度上的神经活动更有意义。本手稿为初学者提供了有关如何制作微型驱动系统并使用通用软件设置参数的信息，以快速直接的方式计算尖峰和LFP信号，以便开始记录和分析。
尽管脑功能的非侵入性记录，例如使用脑电图 （EEG） 和从头皮记录的事件相关电位 （ERP），已广泛用于人类和啮齿动物研究，但 EEG 和 ERP 数据的空间和时间特性较低，因此无法检测特定大脑区域内附近树突突触活动产生的精确信号1.目前，通过利用有意识动物的多通道记录，可以通过在多次行为测试中将微驱动系统植入灵长类动物或啮齿动物的大脑中，长期和渐进地记录大脑深层的神经活动 1,2,3,4,5,6,7,8,9 .简而言之，研究人员可以构建一个微型驱动系统，该系统可用于电极或四极管的独立定位，以靶向大脑的不同部位10,11。例如，Chang等人描述了通过组装轻巧紧凑的微型驱动器12来记录小鼠尖峰和LFP的技术。此外，带有定制附件组件的微机械硅探针已上市，用于在行为任务期间记录啮齿动物的多个单个神经元和 LFP13。尽管已经使用了各种设计来组装微型驱动系统，但就整个微型驱动系统的复杂性和重量而言，这些设计仍然取得了有限的成功。例如，Lansink等人展示了一种具有复杂结构的多通道微驱动系统，用于从单个大脑区域14进行记录。Sato等人报道了一种多通道微型驱动系统，该系统具有自动液压定位功能15。这些微型驱动系统的主要缺点是它们太重，老鼠无法自由移动，并且对于初学者来说很难组装。尽管多通道细胞外记录已被证明是一种在行为测试中测量神经活动的合适且有效的技术，但对于初学者来说，记录和分析复杂的微驱动系统获得的信号并不容易。鉴于在自由移动的小鼠16,17中很难开始多通道细胞外记录和数据分析的整个操作过程，本文提出了简化的指南，以介绍使用常用组件和设置制作微驱动系统的详细过程;此外，还提供了通用软件中用于快速直接计算尖峰和LFP信号的参数。此外，在该协议中，由于使用了氦气球，鼠标可以自由移动，这有助于抵消头部和微型驱动系统的重量。总的来说，在本研究中，我们描述了如何轻松构建微驱动系统并优化记录和数据分析过程。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.54 mm pin header</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>1 x 5</td>
			<td>Applying for the movable micro-drive which can slide on its stulls.</td>
		</tr>
		<tr>
			<td>Adobe Illustrator CC 2017</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>To optimize images from GraphPad.</td>
		</tr>
		<tr>
			<td>BlackRock Microsystems</td>
			<td>Blackrock Neurotech</td>
			<td>Cerebus</td>
			<td>This systems includes headsatge, DA convert, amplifier and computer.</td>
		</tr>
		<tr>
			<td>Brass nut</td>
			<td>Dongguan Gaosi Technology Co., Ltd.</td>
			<td>M0.8 brass nut</td>
			<td>The nut fixes the position of screw.</td>
		</tr>
		<tr>
			<td>Brass screw</td>
			<td>Dongguan Gaosi Technology Co., Ltd.</td>
			<td>M0.8 x 11 mm brass screw</td>
			<td>A screw that hold the movable micro-drive.</td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Guangdong Zhiyuan Biomedical Technology Co., LTD.</td>
			<td>N/A</td>
			<td>12 weeks of age.</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Biosharp</td>
			<td>15 mL; BS-150-M</td>
			<td>To store mice brain with sucrose sulutions.</td>
		</tr>
		<tr>
			<td>Conducting paint</td>
			<td>Structure Probe, Inc.</td>
			<td>7440-22-4</td>
			<td>To improve the lead-connecting quality between connector pins and Ni-wires.</td>
		</tr>
		<tr>
			<td>Conductive copper foil tape</td>
			<td>3M</td>
			<td>1181</td>
			<td>To reduce interferenc.</td>
		</tr>
		<tr>
			<td>Connector</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>2 x 10P</td>
			<td>To connect the headtage to micro-drive system.</td>
		</tr>
		<tr>
			<td>DC Power supply</td>
			<td>Maisheng</td>
			<td>MS-305D</td>
			<td>A power device for&#160; electrolytic lesion.</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>Shanghai New Century Dental Materials Co., Ltd.</td>
			<td>N/A</td>
			<td>To fix the electrode arrays on mouse&#39;s skull after finishing the implantation.</td>
		</tr>
		<tr>
			<td>Digital analog converter</td>
			<td>Blackrock</td>
			<td>128-Channel</td>
			<td>A device that converts digital data into analog signals.</td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>Alteco</td>
			<td>N/A</td>
			<td>To cover pins.</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>To summarize data after analysis.</td>
		</tr>
		<tr>
			<td>Eye scissors</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery or cutting the Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>JiangXi YuYuan Medical Equipment Co.,Ltd.</td>
			<td>N/A</td>
			<td>For surgery or assembling the mirco-drive system.</td>
		</tr>
		<tr>
			<td>Freezing microtome</td>
			<td>Leica</td>
			<td>CM3050 S</td>
			<td>&#160;Cut the mouse&#8217;s brain into slices</td>
		</tr>
		<tr>
			<td>Fused silica capillary tubing</td>
			<td>Zhengzhou INNOSEP Scientific Co., Ltd.</td>
			<td>TSP050125</td>
			<td>To&#160; serve as the guide tubes for Ni-chrome wires.</td>
		</tr>
		<tr>
			<td>Glass microelectrode</td>
			<td>Sutter Instrument Company</td>
			<td>BF100-50-10</td>
			<td>To mark the desired locations for implantation using the filled ink.</td>
		</tr>
		<tr>
			<td>GraphPad Prism 7</td>
			<td>GraphPad Software</td>
			<td>N/A</td>
			<td>To analyze and visualize the results.</td>
		</tr>
		<tr>
			<td>Guide-tube</td>
			<td>Polymicro technologies</td>
			<td>1068150020</td>
			<td>To load Ni-chrome wires.</td>
		</tr>
		<tr>
			<td>Headstage</td>
			<td>Blackrock</td>
			<td>N/A</td>
			<td>A tool of transmitting signals.</td>
		</tr>
		<tr>
			<td>Helium balloon</td>
			<td>Yili Festive products Co., Ltd.</td>
			<td>24 inch</td>
			<td>To offset the weight of headstage and micro-drive system.</td>
		</tr>
		<tr>
			<td>Ink</td>
			<td>Sailor Pen Co.,LTD.</td>
			<td>13-2001</td>
			<td>To mark the desired locations for implantation.</td>
		</tr>
		<tr>
			<td>Iodine tincture</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To disinfect mouse&#39;s scalp.</td>
		</tr>
		<tr>
			<td>Lincomycin in Hydrochloride and Lidocaine&#160; hydrochloride gel</td>
			<td>Hubei kangzheng pharmaceutical co., ltd.</td>
			<td>10g</td>
			<td>A drug used to reduce inflammation.</td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Vicki Biotechnology Co., Ltd.</td>
			<td>71125-38-7</td>
			<td>To reduce postoperative pain in mice.</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Scientifica</td>
			<td>Scientifica IVM Triple</td>
			<td>For electrode arrays implantation.</td>
		</tr>
		<tr>
			<td>Microscope&#160;</td>
			<td>Nikon</td>
			<td>ECLIPSE Ni-E</td>
			<td>&#160;Capture the images of brain sections</td>
		</tr>
		<tr>
			<td>nanoZ impedance tester</td>
			<td>Plexon</td>
			<td>nanoZ</td>
			<td>To measure impedance or performing electrode impedance spectroscopy (EIS) for multichannel microelectrode arrays.</td>
		</tr>
		<tr>
			<td>NeuroExplorer</td>
			<td>Plexon</td>
			<td>NeuroExplorer</td>
			<td>A tool for analyzing the electrophysiological data.</td>
		</tr>
		<tr>
			<td>NeuroExplorer&#160;</td>
			<td>Plexon, USA</td>
			<td>N/A</td>
			<td>A software.</td>
		</tr>
		<tr>
			<td>Ni-chrome wire</td>
			<td>California Fine Wire Co.</td>
			<td>M472490</td>
			<td>35 &#956;m Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Offline Sorter</td>
			<td>Plexon</td>
			<td>Offline Sorter</td>
			<td>A tool for sorting the recorded multi-units.</td>
		</tr>
		<tr>
			<td>PCB board</td>
			<td>Hangzhou Jiepei Information Technology Co., Ltd.</td>
			<td>N/A</td>
			<td>Computer designed board.</td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma</td>
			<td>P3761</td>
			<td>To anesthetize mice.</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Sigma</td>
			<td>57-33-0</td>
			<td>To anesthetize the mouse.</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Longer</td>
			<td>BT100-1F</td>
			<td>A device used for perfusion</td>
		</tr>
		<tr>
			<td>Polyformaldehyde&#160;</td>
			<td>Sangon Biotech</td>
			<td>A500684-0500</td>
			<td>The main component of fixative solution for fixation of mouse brains&#160;</td>
		</tr>
		<tr>
			<td>PtCl4</td>
			<td>Tianjin Jinbolan Fine Chemical Co., Ltd.</td>
			<td>13454-96-1</td>
			<td>Preparation for gold plating liquid.</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To clean the mouse&#39;s skull.</td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Suzhou Xinye Electronics Co., Ltd.</td>
			<td>2 mm diameter</td>
			<td>Applying for ground and reference electrodes.</td>
		</tr>
		<tr>
			<td>Skull drill</td>
			<td>RWD Life Science</td>
			<td>78001</td>
			<td>To drill carefully two small holes on mouse&#39;s skull.</td>
		</tr>
		<tr>
			<td>Stainless steel screws</td>
			<td>YOUXIN Electronic Co., Ltd.</td>
			<td>M0.8 x 2</td>
			<td>To protect the micro-drive system and link the ground and reference electrodes.</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>RWD Life Science</td>
			<td>68513</td>
			<td>To perform the stereotaxic coordinates of bilateral motor cortex.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Damao</td>
			<td>57-50-1</td>
			<td>To dehydrate the mouse brains&#160; after perfusion.</td>
		</tr>
		<tr>
			<td>Super glue</td>
			<td>Henkel AG &#38; Co.</td>
			<td>PSK5C</td>
			<td>To fix the guide tube and Ni-chrome wire.</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Harvard Apparatus</td>
			<td>TCAT-2</td>
			<td>To maintain mouse&#39;s rectal temperature at 37&#176;C</td>
		</tr>
		<tr>
			<td>Tetracycline eye ointment</td>
			<td>Guangdong Hengjian Pharmaceutical Co., Ltd.</td>
			<td>N/A</td>
			<td>To protect the mouse&#39;s eyes during surgery.</td>
		</tr>
		<tr>
			<td>Thread</td>
			<td>Rapala</td>
			<td>N/A</td>
			<td>To link ballon and headstage.</td>
		</tr>
		<tr>
			<td>Vaseline</td>
			<td>Unilever plc</td>
			<td>N/A</td>
			<td>To cover the gap between electrode arrays and mouse&#39;s skull.</td>
		</tr>
	</tbody>
</table>

multichannel extracellular recording in freely moving mice

该协议旨在通过将电生理信号与自发和/或特定行为相关联来揭示神经元放电和网络局部场电位（LFP）在执行特定任务的行为小鼠中的特性。该技术代表了研究这些行为背后的神经元网络活动的宝贵工具。本文提供了在自由移动的清醒小鼠中进行电极植入和随后的细胞外记录的详细而完整的程序。该研究包括植入微电极阵列的详细方法，使用多通道系统捕获运动皮层 （MC） 中的 LFP 和神经元脉冲信号，以及随后的离线数据分析。在有意识的动物中多通道记录的优点是可以获得和比较更多脉冲神经元和神经元亚型，从而可以评估特定行为与相关电生理信号之间的关系。值得注意的是，本研究中描述的多通道细胞外记录技术和数据分析程序在行为小鼠中进行实验时可以应用于其他大脑区域。

应用高通（250 Hz）滤波器从原始信号中提取多单元尖峰（图6A）。此外，验证了通过PCA排序的正常小鼠MC的记录单位（图7A-D），并记录了小鼠MC中单位的谷值宽度和波形持续时间。结果表明，小鼠MC推定锥体神经元（Pyn）的谷宽和波形持续时间均高于推定中间神经元（IN）（图7E，F;两个样本Mann-Whitney检验;?...

Watch this Scientific Journal Video about Advancements in Multichannel Extracellular Recording for Studying Neuronal Activity in Freely Moving Mice at JoVE.com

作者聚焦:用于研究自由移动小鼠神经元活动的多通道细胞外记录的进展 

该协议描述了运动皮层（MC）中细胞外记录的方法，以揭示自由移动的有意识小鼠的细胞外电生理特性，以及局部场电位（LFP）和尖峰的数据分析，这对于评估网络神经活动的潜在行为是有用的。

自由移动小鼠的多通道细胞外记录

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by ...

我们旨在通过将电生理信号与行为相关联来揭示行为小鼠执行特定任务的神经元放电和网络局部场电位的特性。微驱动系统的多通道细胞外记录已被证明是行为测试期间神经活动的合适且有效的技术。在自由移动的小鼠中进行多通道记录被认为是神经科学研究中的一项有用技术，但对于初学者来说，获取和分析信号仍然相当具有挑战性。我们介绍了如何利用更稳定、更轻便的微驱动系统对自由移动的小鼠进行多通道细胞外记录，并针对初学者优化了记录和数据分析的过程。我们将在新版本中增加通道数并减少微型驱动器系统的音量。

multichannel-extracellular-recording-in-freely-moving-mice

Research

JoVE Journal

Neuroscience

Multichannel Extracellular Recording in Freely Moving Mice

2.5K 次观看.  South China Normal University. 我们旨在通过将电生理信号与行为相关联来揭示行为小鼠执行特定任务的神经元放电和网络局部场电位的特性。微驱动系统的多通道细胞外记录已被证明是行为测试期间神经活动的合适且有效的技术。在自由移动的小鼠中进行多通道记录被认为是神经科学研究中的一项有用技术，但对于初学者来说，获取和分析信号仍然相当具有挑战性。我们介绍了如何利用更稳定、更?...

视频: 作者聚焦:用于研究自由移动小鼠神经元活动的多通道细胞外记录的进展 

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

高密度的自由移动，使用基于聚酰亚胺的微电极的小鼠的脑电图记录

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

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 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 ...

Extracellular Wire Tetrode Recording in Brain of Freely Walking Insects

Increasing interest in the role of brain activity in insect motor control requires that we be able to monitor neural activity while insects perform natural behavior. We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allowed us to record activity from multiple neurons simultaneously while a tethered cockroach turned or altered walking speed. While a major advance, tethered preparations provide access to limited behaviors and often lack feedback processes that occur in freely moving animals. We now present a modified version of that technique that allows us to record from the central complex of freely moving cockroaches as they walk in an arena and deal with barriers by turning, climbing or tunneling. Coupled with high speed video and cluster cutting, we can now relate brain activity to various parameters of the movement of freely behaving insects.

We previously developed a technique for implanting tetrode wires into the central complex of cockroach brains that allows us to monitor activity in individual units of tethered cockroaches. Here we present a modified version of that technique that allows us to also record brain activity in freely moving insects.

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 Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

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 ...

Noninvasive EEG Recordings from Freely Moving Piglets

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel telemetric electroencephalography system in combination with standard self-adhesive hydrogel electrodes. The piglets are calmed down without the use of sedatives. After their release into the pigpen, the piglets behave normally&#8212;they drink and sleep in the same cycle as their siblings. Their sleep phases are used for the EEG recordings.

Here, we present a protocol to record telemetric electroencephalograms (EEGs) from freely moving piglets directly in the pigpen without the use of a sedative, making it possible to record typical EEG patterns during non-REM sleep, like spindle bursts.

自由移动仔猪的无创脑电图记录

The method allows the recording of high-quality electroencephalograms (EEGs) from freely moving piglets directly in the pigpen. We use a one-channel ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

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, ...