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。

The local field potential (LFP), an important component of extracellular signals, reflects the synaptic activity of large populations of neurons, which form the neural code for multiple behaviors1. Spikes generated by neuronal activity are considered to contribute to the LFP and are important for neural coding2. Alterations in spikes and LFPs have been proven to mediate several brain diseases, such as Alzheimer&#39;s disease, as well as emotions such as fear, etc.3,4. It is worth noting that many studies have highlighted that spike activity significantly differs between awake and anesthetized states in animals5. Although recordings in anesthetized animals offer an opportunity to assess LFPs with minimal artifacts in highly defined cortical synchronization states, the results differ to some extent from what can be found in awake subjects6,7,8. Thus, it is more meaningful to detect neural activity over long time scales and large spatial scales in various diseases in an awake brain state using electrodes implanted in the brain. This manuscript provides information for beginners on how to make the micro-drive system and set the parameters using common software for computing the spike and LFP signals in a fast and straightforward way in order to get the recording and analysis started.
Although the non-invasive recording of brain functions, such as by using electroencephalograms (EEGs) and event-related potentials (ERPs) recorded from the scalp, has been used extensively in human and rodent studies, EEG and ERP data have low spatial and temporal properties and, thus, cannot detect the precise signals produced by nearby dendritic synaptic activity within a specific brain area1. Currently, by taking advantage of multichannel recording in conscious animals, neural activity in the deeper layers of the brain can be recorded chronically and progressively by implanting a micro-drive system into the brains of primates or rodents during multiple behavioral tests1,2,3,4,5,6,7,8,9. Briefly, researchers can construct a micro-drive system that can be used for the independent positioning of the electrodes or tetrodes to target different parts of the brain10,11. For example, Chang et al. described techniques to record spikes and LFPs in mice by assembling a light and compact micro-drive12. In addition, micro-machined silicon probes with custom-made accessory components are commercially available for recording multiple single neurons and LFPs in rodents during behavioral tasks13. Although various designs have been used for assembling micro-drive systems, these still have limited success in terms of the complexity and weight of the whole micro-drive system. For example, Lansink et al. showed a multichannel micro-drive system with a complex structure for recording from a single brain region14. Sato et al. reported a multichannel micro-drive system displaying an automatic hydraulic positioning function15. The main disadvantages of these micro-drive systems are that they are too heavy for mice to move freely and are difficult to assemble for beginners. Although multichannel extracellular recording has been shown to be a suitable and efficient technology for measuring neural activity during behavioral tests, it is not easy for beginners to record and analyze the signals acquired by the complex micro-drive system. Given that it is difficult to get the entire operation process of the multichannel extracellular recording and data analysis started in freely moving mice16,17, this present article presents simplified guidelines to introduce the detailed process of making the micro-drive system using commonly available components and settings; the parameters in the common software for computing the spike and LFP signals in a fast and straightforward way are also provided. Additionally, in this protocol, the mouse can move freely due to the use of a helium balloon, which contributes to offsetting the weight of headstage and micro-drive system. Generally, in the present study, we describe how to easily build a micro-drive system and optimize the processes of recording and data analysis.

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 

自由移动小鼠的多通道细胞外记录

We aim to uncover the properties of neuronal firing and network local field potentials in behaving mice carrying out specific tasks by correlating the electrophysiological signal with behaviors. Multichannel extracellular recording by the micro-drive system has been shown to be a suitable and a efficient technology for neural activity during behavioral tests. Multichannel recording in free-moving mice has been deemed to be a useful technology in neuroscience studies, but it's still quite challenging for beginners to acquire and analyze the signals.We introduced how to perform the multichannel extracellular recording in freely-moving mice with a more steady and lightweight micro-drive system, and optimize the processes of recording and data analysis for the beginners. We are going to increase the number of channels and reduce the volume of micro-driver system in a new version.

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

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。

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

1.9K 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 

The protocol aims to uncover the properties of neuronal firing and network local field potentials (LFPs) in behaving mice carrying out specific tasks by correlating the electrophysiological signals with spontaneous and/or specific behavior. This technique represents a valuable tool in studying the neuronal network activity underlying these behaviors. The article provides a detailed and complete procedure for electrode implantation and consequent extracellular recording in free-moving conscious mice. The study includes a detailed method for implanting the microelectrode arrays, capturing the LFP and neuronal spiking signals in the motor cortex (MC) using a multichannel system, and the subsequent offline data analysis. The advantage of multichannel recording in conscious animals is that a greater number of spiking neurons and neuronal subtypes can be obtained and compared, which allows the evaluation of the relationship between a specific behavior and the associated electrophysiological signals. Notably, the multichannel extracellular recording technique and the data analysis procedure described in the present study can be applied to other brain areas when conducting experiments in behaving mice.

The protocol describes the methodology of extracellular recording in the motor cortex (MC) to reveal extracellular electrophysiological properties in freely moving conscious mice, as well as the data analysis of local field potentials (LFPs) and spikes, which is useful for evaluating the network neural activity underlying behaviors of interest.

科研

神经科学

Micro-Drive System Assembly and Electrode Array Implantation for Multichannel Recording in Mice

Begin by assembling the Microdrive system. Connect two computer-designed boards using two stulls and a screw that holds the movable Microdrive, and attach the connector to one board. Ensure the Microdrive can carry two sets of eight guide tubes for each side of the motor cortex or MC region.Cut the guide tubes to the same length. Cut 16 nichrome wires, each measuring approximately five centimeters long and 35 micrometers in diameter. Load the wires into the guide tubes and apply glue to fix them.Strip the wire insulation and twine each exposed wire to each pin from the connector following the channel map and the reference and ground electrodes, then slowly coat conducting paint onto each pin. Cover the pins using epoxy resin, then perform gold plating via an impedance tester to reduce the impedance of the electrode tips to approximately 350 kiloohms. For electrode array implantation, fix the anesthetized mouse in a stereotaxic apparatus and use a temperature controller to maintain its rectal temperature at 37 degrees Celsius.Then make a small midline incision to expose the skull. Remove the residual tissue using scissors and clean the skull with sterile saline-soaked cotton buds. Using a glass microelectrode filled with ink, mark the desired locations of the bilateral MC for implantation.Next, using a skull drill, carefully drill two small holes on both the left and right sides of the coordinated skull in the MC regions. Gently remove the dura mater from the holes with fine forceps. Then insert the Microdrive system into the center of the holes, using a micromanipulator at 10 micrometers per second.After finishing the insertion, fill the petroleum jelly into the dental cement walls and join the bottom plate of the Microdrive system and the dental cement walls with the mixed dental cement.

用于小鼠多通道记录的微驱动系统组装和电极阵列植入

Multichannel Recording in the Bilateral MC in Free-Moving Mice and Neurophysiological Data Analysis

For the multi-channel recording, move down the electrode arrays by twisting the screw on the movable part of the micro-drive system one day in advance. Hold the head of an awake mouse lightly and carefully. Then, link the center of the head stage and a helium balloon with a thread to offset the weight of the head stage and the micro-drive system.Capture raw signals using the recording electrodes and multi-channel systems by sampling at 30 kilohertz in the recording software. Then digitize using a digital analog converter from the multi-channel systems. Extract the local field potential, or LFP signals, from the raw data by re-sampling at 10 kilohertz.Then, using a notch filter, remove the 50-hertz line noise. For spike sorting and analysis, in the spike sorting software, click on File, then Open, and nev files to open the spike data sampled at 30 kilohertz. Click on Info to select the unsorted channel.Then choose Sort, Change Sort Method, and Use K-Means. Press the button Valley Seeking Sort, then K-Means Sorting to obtain the sorted units. Next, in the neurophysiological data analysis software, open the sorted spike file by clicking on File, Import Data, and BlackRock File.To obtain the autocorrelogram for the selected unit, click on Analysis, then Autocorrelograms, and set the parameters. Load the sorted spike data. Then, click on Analysis, and Interspike Interval Histograms to obtain the interspike interval histogram, and then set the desired parameters.Click on Analysis, then Crosscorrelograms to obtain the crosscorrelogram between two sorted unit events. And then set the reference events and parameters. For LFP analysis, click on File, Import Data, and BlackRock File, to open the continuous signal data sampled at 10 kilohertz.Then, by clicking Analysis and Spectrum for Continuous, analyze the power spectrum for the LFP from the selected channel. Next, click on Analysis, then Coherence for Continuous to analyze the coherence for two LFPs from the left and right sides of the MC.Then, click on Analysis, followed by Correlation with Continuous Variables to analyze the correlation between two LFPs from the left and right sides of the MC.Once done, click on Results, then Numerical Results to save the Power Spectrum Density, Coherence, and Correlation with a xls file name extension. To analyze correlations between the spike and LFP, click on File, then Import Data and BlackRock File to open the continuous signal data and spike data.Then, click on Analysis and Coherence Analysis to analyze the coherence between the spikes and LFP from the selected channel. Finally, click on Results, then Numerical Results to save the results of the spike field coherence with a xls file name extension. The valley width and waveform duration of the units in the MC of the mouse showed that both the valley width and waveform duration of the MC putative pyramidal neurons in mice are higher than those of the putative interneurons.The crosscorrelogram between putative pyramidal neurons and interneurons indicated that the putative pyramidal neurons spiking occurs before the putative interneurons with a window of 18 milliseconds. In the LFP analysis, the LFPs of the left and right MC in normal mice were similar in the power spectrum, suggesting synchronized activities between the left and right MC.Further, the coherence and correlation between the left and right MC were calculated. The curve of spike field coherence in the MC of a normal mouse showed a stronger low gamma coherence for the putative interneurons compared to the pyramidal neurons.