我们要感谢米格尔·布尔戈斯先生和古斯塔沃·拉戈先生提供技术援助。我们感谢Stoelting公司支付录像制作费用，感谢Jinga-hi公司提供技术援助，感谢墨西哥伊比利亚美洲大学调查和调查部为这项工作提供资金。

所有程序均遵循国家卫生机构和墨西哥当地法律实施的《实验动物护理和使用指南》（NIH出版物第8023号，1978年修订），以减少用于动物福利的动物数量并禁止动物痛苦。伊比利亚美洲大学伦理委员会批准了在这项研究中使用动物的实验方案。
1. 常规设置
<ol><li>根据制造说明在计算机上安装行为跟踪软件。</li>
	<li>将相机直接安装在设备上方，使其朝下。相机应连接到计算机。</li>
	<li>安装相机所需的驱动程序软件（按照制造说明进行操作）。</li>
	<li>如果相机包含变焦镜头，请调整它们以完全适合相机的显示屏。</li>
	<li>按照制造软件关闭相机自动对焦（AF）模式。</li>
	<li>确保摄像机实时正常工作，并测试视频捕获模式，直到可以使用为止。</li>
</ol>2. 环境富集方案（见图1）
注意:三个月大的雄性Wistar大鼠用于本实验，并在自然暗光条件下保持。
<ol><li>将锯末床上用品放置在透明的亚克力方形竞技场（500 x 500 x 500 mm）中。</li>
	<li>在竞技场上放置三种不同类型的玩具，供啮齿动物互动（例如，活动轮、双层甲板、楼梯等）。</li>
	<li>添加四个 2 英寸和四个弯曲的灰色不透明 PVC 管。</li>
	<li>为食物和饮水机提供 随意接触动物 的机会。</li>
	<li>在常规条件下，在动物饲养室内，每个笼子放置三只啮齿动物。</li>
	<li>根据相应的协议，将动物留在该竞技场所需的时间。在这个实验中，动物应该在竞技场内停留20天。 注意:电极植入手术后，动物不会回到环境富集治疗。相反，它们被放在单个笼子里，直到新的物体识别测试完成。</li>
</ol>3. 电极制造工艺
<ol><li>在大约 2 厘米处切割一根铜线，然后用砂纸从两端擦拭约 0.5 厘米。</li>
	<li>将铜线的一端滚动到小尺寸螺钉（电极）的头部，并确保其牢固固定，因为这是一个关键步骤。必须保证两种材料之间的正确接触，以避免脑电信号中的伪影。</li>
	<li>将另一端插入连接器的端子尖端，并确保使用细镊子加固来正确固定。此尖端应与放大器电缆连接。</li>
	<li>使用万用表测量从尖端到螺杆的合适电导率。此过程可确保正确安装电极连接。</li>
</ol>4.硬膜外（颅骨螺钉）电极植入手术
注意:经过20天的环境富集治疗后，动物将按照以下描述的程序进行手术:
<ol><li>向动物注射氯胺酮/甲苯噻嗪（90/10毫克/千克，腹腔注射）的混合物。 注意:为避免气道阻塞，请等到大鼠停止移动，然后将其从笼子中取出并将动物放在平坦的表面上。注射非甾体抗炎药（美洛昔康 1 mg/kg，皮下）和抗生素（恩诺沙星 2.5 mg/kg，口服）作为先发制人的镇痛药。</li>
	<li>一旦大鼠完全麻醉，剃掉大鼠的头部区域。 注意:在继续手术之前，请确保动物已完全麻醉。小心地捏住其中一条腿或尾巴。如果动物对刺激有反应，再等几分钟，然后再次捏住它。如果动物对捏没有反应，请转到下一步。如果有所需的设备，强烈建议使用气体麻醉（如异氟醚），因为为了安全起见，它更容易滴定。</li>
	<li>首先用耳杆固定双耳，将动物放在立体定位装置上（注意不要伤害动物的内耳）。最后，将门牙放在咬杆上并固定鼻杆。 注意:为所有手术为动物提供加热垫，因为此手术中使用的麻醉通常会导致体温过低和呼吸问题。</li>
	<li>使用三轮交替的洗必泰或碘基磨砂膏清洁头部区域顶部，然后用生理盐水或酒精冲洗。</li>
	<li>在头部区域（0.5 mL）皮肤下皮下注射利多卡因（20 mg/mL）。</li>
	<li>每5-10分钟向每只动物的眼睛滴一滴眼药溶液或盐水，以帮助它们不干燥。</li>
	<li>使用手术刀，从前向到后方向切开约 2 厘米的切口，以正确暴露颅骨的顶部区域。</li>
	<li>使用斗牛犬夹子缩回皮肤并刮擦覆盖在颅骨上的组织。</li>
	<li>识别并记录获得的布雷格玛坐标。</li>
	<li>从布雷格玛开始，使用立体定位Paxinos和Watson Atlas30，定位并标记电极将固定的七个点（坐标）中的每一个的位置。 注意:在这个实验中，F3，F4螺钉（+距布雷格玛2.0毫米，距中线2.25毫米）;C3，C4螺钉（距布雷格玛-3.0毫米，距中线2.75毫米）;和 P3，P4 螺钉（距布雷格玛 −7.0 毫米，距中线 2.75 毫米横向）。第七颗螺钉位于鼻骨（NZ）的后方，作为地面参考（见 图2）。</li>
	<li>使用变速钻具，在每个标记上打一个尖端尺寸为 2（长度 44.5 毫米）的孔，注意不要完全穿透头骨。</li>
	<li>将电极插入孔中并小心地将其拧入颅骨。</li>
	<li>重复步骤 4.10 和 4.11，直到所有七个螺钉都正确固定。</li>
	<li>用第一层牙科水泥固定所有 7 个螺钉。将每个电极插入连接器。用第二层牙科水泥（这将防止动物拉下螺丝）和连接器底部完全覆盖电线。如有必要，用第三层牙科水泥覆盖，保持脑电图连接器清洁以进行正确连接，以便可以正确连接脑电图设备（见 图3）。 注意:放置每对双边螺钉后，可以用牙科水泥固定这些螺钉（可选步骤）。</li>
	<li>将大鼠留在术后护理中过夜。观察动物并在手术后为动物提供加热垫1-2小时，因为该程序中使用的麻醉通常会导致体温过低和呼吸问题。</li>
	<li>皮下注射50mL/kg/24h（维持剂量）盐水溶液，以防止脱水。手术后和接下来的24小时内注射非甾体抗炎药（美洛昔康2mg / kg，皮下）和抗生素（恩诺沙星5mg / kg，口服）。</li>
	<li>手术后，在进行行为测试之前，将大鼠放在单笼中以在七天内完全恢复。</li>
	<li>定期（至少每天一次）轻轻地操纵动物，以帮助减轻未来操作中的压力。在用一只手握住大鼠的同时，手指轻轻地施加在动物的背部，将手指滑过皮毛。手术后一周检查头部伤口、健康状况、一般行为和体重。 注意:如果在动物身上发现任何异常或疾病/压力的迹象，请通知负责的兽医。在此期间之后，执行新型物体识别测试和脑电图记录技术。</li>
</ol>5. 新型物体识别测试
注意:手术后七天，进行行为测试。在所提出的实验中，所有行为程序都在14小时00分钟和16小时00分钟之间进行，这对应于大鼠的光周期。
<ol><li>将一件由柔软织物制成的背心（在行为测试期间将脑电图装置放置在该背心上）放在大鼠身上。在进行行为测试之前，允许习惯2-3天。</li>
	<li>将黑色亚克力方形竞技场（500 x 500 x 500 mm）放置在昏暗照明的录音室中。</li>
	<li>使用双面胶带将两个相同的新物体固定在竞技场的地板中心（以防止其被动物移位）。物体之间和竞技场墙壁必须等距。</li>
	<li>事先用 50% 乙醇彻底清洁每个物体，并在每次试验后彻底清洁竞技场的地板（以避免嗅觉提示）。 注意:始终在每次会议开始前至少半小时将动物转移到宿舍（从动物饲养室到实验室）。完成记录后，将动物留在实验室中再呆一个小时。这是为了避免可能影响此测试性能的压力。</li>
	<li>在开始每次测试之前连接脑电图设备。轻轻约束动物，并将电缆牢固地插入动物头部的连接器，并将脑电图套件连接到动物的背部（见 图4）。只允许一个职位。 注意:先前对动物的温和操作可能有助于减轻连接过程中动物的压力。否则，设备或动物损坏的风险会增加。使用 USB 端口为设备电池充满电。</li>
	<li>新型物体识别测试阶段<ol><li>习惯性:连续两天每隔5分钟处理一次动物，然后立即将动物放在竞技场上（没有任何物体），让它们自由探索10分钟。 注意:在执行任何采集和记忆测试会话之前，小心处理大鼠并将其连接到相应的EEG设备，该设备在开始测试之前已正确固定。</li>
		<li>获取环节:将动物放在竞技场上，面向与物体相对的一面墙。让动物自由探索10分钟。转到步骤 6.13 以使用行为跟踪软件进行测试记录。 注意:确保脑电图装置正确固定附着在大鼠背部的背心（以确保正确跟踪动物）。如需额外加固，请使用遮蔽胶带。</li>
		<li>短期记忆测试（SMT）:用形状，颜色和纹理完全不同的任何其他物体替换其中一个物体。在采集过程后2小时，将动物放在面向物体对面的墙壁之一的竞技场中。让动物自由探索10分钟。转到步骤 6.13 以使用行为跟踪软件进行测试记录。</li>
		<li>长期记忆测试（LMT）:用与短期记忆测试在形状，颜色和纹理上完全不同的任何其他物体替换所使用的物体。在采集过程后24小时将动物放置在面向物体对面的墙壁之一的竞技场中。让动物自由探索 10 分钟 转到步骤 6.13 使用行为跟踪软件进行测试记录。</li>
	</ol></li>
</ol>6. 行为跟踪软件设置
<ol><li>打开行为跟踪软件。</li>
	<li>使用机构的用户和密码登录帐户。</li>
	<li>打开点击"新建空实验"，然后选择实验方案的名称（例如，"NORT"）。</li>
	<li>选择"视频跟踪模式"。 注意:在此实验中，摄像机设置为实时流式传输视频跟踪。但是，还有一个选项可以选择预先录制的视频。</li>
	<li>转到"设备"。通过将橙色矩形调整到投影竞技场的极限来定义竞技场区域。确定对象的区域，在屏幕上的摄像机投射的竞技场内对象边界处拟合橙色圆圈。</li>
	<li>将比例移动标尺线设置到沿图像（竞技场）已知长度的位置。在"设置"面板上的"标尺线长度为"选项中输入对象的长度（以毫米为单位）。在这种情况下，竞技场的尺寸为 500 x 500 毫米。</li>
	<li>转到"跟踪和行为"。继续"区域"。单击"添加项目"菜单，然后选择"新区域"。选择竞技场区域并命名新区域（例如，"场地"）。</li>
	<li>对对象的区域重复上一步并命名新区域（例如，"对象"）。</li>
	<li>转到"动物颜色"并选择"动物比设备背景浅"选项。 注意:白色（Wistar）大鼠用于本实验。但是，该软件为使用黑鼠和斑点大鼠的研究人员提供了其他选择。两种动物都可以在同一实验中使用。</li>
	<li>转到"跟踪动物的头部和尾巴"，然后选择"是的，我希望跟踪动物的头部和尾巴"。</li>
	<li>转到"测试" |"阶段"，然后从菜单"添加项目"中选择"新建阶段"。将新阶段命名为"收购"。定义阶段的持续时间（例如，600 秒）。</li>
	<li>重复"短期记忆测试"和"长期记忆测试"阶段的上一步。 注意:在此协议中，所有阶段具有相同的持续时间（10分钟）。</li>
	<li>转到"程序"。定义每个阶段（采集、短期记忆测试和长期记忆测试）要跟踪的事件。</li>
	<li>开始测试（每只动物）。转到"测试"（在顶部菜单栏中），然后选择"添加测试 （+）"。为要测试的动物分配一个编号（例如，"1"）。</li>
	<li>选择"记录"并命名动物和会话（例如，"M1 Acq"）。</li>
	<li>在将动物放入竞技场之前，请在"播放"按钮上单击一次。将显示一条消息"等待启动"。</li>
	<li>将动物放入竞技场后，再次单击"播放"按钮。测试将自动开始和结束。</li>
	<li>对短期记忆测试（采集后2小时）和长期记忆测试（采集后24小时）重复步骤6.13-6.16。</li>
</ol>7. 无线电生理设备设置
<ol><li>将调制解调器连接到 PC 主机并将其打开。关闭 PC 上的任何其他网络设备。最好将登记室中的任何其他无线通信静音，例如蓝牙、手机、其他调制解调器甚至无线手机。</li>
	<li>将放大器连接到大鼠的背部，如步骤5.5中所述。</li>
	<li>通过插入电池打开脑电图设备。 注:连接设备2秒后，EEG放大器上的红色LED将闪烁，表示与调制解调器的通信处于活动状态，然后绿色LED将亮起。如果通信成功，调制解调器上的指示灯将开始持续闪烁。放大器现在已准备好向调制解调器发送信息。</li>
	<li>启动脑电图软件并根据制造商的说明进行设置，以集成到无线脑电图采集设备中</li>
	<li>按"开始显示"按钮。EEG软件将显示实际的信号采集。 注意:使用"Windows 任务管理器"分配"实时"优先级模式，以避免在实验过程中丢失信息。</li>
</ol>8. 脑电图（EEG）信号记录
<ol><li>在验证脑电图软件正在获取数据后，启动行为跟踪软件，并设置实验方案以验证动物是否在观察区并且设置是否正常工作。</li>
	<li>此时，按"开始记录"按钮开始脑电图软件记录。检查采集信号是否正在运行后，在BTS中开始实验。</li>
	<li>实验结束后，返回脑电图软件并停止记录过程。记录将使用默认名称保存，该名称由使用以下格式的录制日期组成:"yyyy-mmdd-hhmm_SubjectID_Ephys.plx"。默认情况下，所有记录都保存在脑电图软件（神经物理学数据）文件夹中。</li>
	<li>检查是否已创建两个数据文件。记录实验日志或更改名称以避免混淆。</li>
</ol>9. 行为任务与脑电信号同步
<ol><li>打开 MATLAB 并执行命令:convert_plx2mat。这样的功能将打开一个浏览器框。转换函数由制造商提供，必须添加到 MATLAB 的路径中。</li>
	<li>选择要转换的 *.plx，然后在 MATLAB 的命令行中按"Enter"将其转换为默认参数。</li>
	<li>打开 BTS 实验文件并转到"协议"。单击选项"结果，报告和数据"选择两个对象的所有事件，然后单击"选择报告的时间格式"，选择第三个选项:"在HH:MM:SS.SS中将事件时间显示为实时 - 例如13:20:14.791。</li>
	<li>现在转到"文件"，然后单击"导出"和"将实验导出为XML"，选中"测试日期和时间"，最后单击"创建XML"。</li>
	<li>转到"导出测试数据"，然后单击"保存数据"。将创建一个包含事件时间的.csv文件。</li>
	<li>对每个文件重复步骤 9.1 到 9.5。在我们的例子中，三个实验是:ACQ，STM和LTM。</li>
	<li>转换 EEG 和行为文件后，将它们收集到单个文件夹中。该文件夹必须有六个文件，分别是三个 .mat 文件和三个.csv。在我们的例子中，文件被称为:PID_01_ACQ_N.mat，PID_02_STM_N.mat，PID_03_LTM_N.mat，PID_01_ACQ_M.csv，PID_02_STM_M.csv和PID_03_LTM_M.csv。 ID是指动物的识别号码。</li>
	<li>使用MATLAB打开"procesa_sujeto.m"功能，并将第二行调整为动物的ID。</li>
	<li>现在将 MATLAB 移动到这样的文件夹并执行:"procesa_sujeto"以创建与 ACQ、STM 和 LTM 平台上的对象识别相关的 alpha 和 beta 相对带与功率相关的图形。 注意:"procesa_sujeto"是执行/运行多个信号处理分析的函数。这些分析总结如下，步骤9.10至9.15。</li>
	<li>使用相位校正，用 [5-40] Hz 的 4 阶巴特沃兹带通滤波器过滤每个 EEG 信号。</li>
	<li>在进行后续分析之前目视检查信号，并且由于电极放置缺陷或动物运动调整错误而导致的伪影通道被排除在进一步分析之外。</li>
	<li>参考公共平均值信号以减轻运动伪影。</li>
	<li>分割脑电图信号以形成由BTS派生的时间戳同步的4秒长度的时期。目标事件是对物体的探索，该物体以动物到物体边界的距离为标志。这些事件标记在 BTS 时间戳上，并用作固定窗口位置的标识符。因此，EEG时期由探索开始前的1秒到之后的3秒来界定。在这一点上，没有使用关于探索长度的验证，但将考虑用于未来的研究。</li>
	<li>使用韦尔奇周期图方法估计这些时期的光谱功率密度，使用1 s窗口长度，重叠90%，傅里叶变换估计之前的汉宁窗口，使用这些参数实现了1 Hz的分辨率。</li>
	<li>通过评估周期图下的面积来评估每个波段的功率谱，给出的值对应于相对能量，这意味着每个脑电波段的能量除以纪元的总能量。此过程还可以减少由于EEG信号伪影而导致的错误估计。</li>
</ol>

<ol>
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Sylvia Ortega-Martinez博士是Stoelting Co.的员工，该公司提供并赞助了本文的制作和开放访问。

行为和脑电图研究本质上是困难和具有挑战性的。因此，这两种技术的结合提出了重要的关键步骤。因此，这两种并发技术都没有被广泛使用。在实际实践中，世界各地的每个小组都在特殊条件下进行行为测试，例如动物、分析参数或治疗。上述情况在该领域引起了重大争议，需要制定每个人都可以使用的标准程序。在这里，我们准备了这个详细的程序，其中包含大多数已发表的文章中通常没有描述或提及的所有关键步骤和方法论考虑因素。这些将在下面讨论。
所需材料的生产是该技术成功的基本步骤。在这方面，需要使用不锈钢螺钉、铜电缆和银焊机从头开始构建电极。这些材料很难永久焊接在一起，因此必须在使用前验证每个电极的导电性和强度。可以使用另一种类型的电线进行电极组件;然而，铜足够灵活，可以操纵电极将其插入放大器连接器。在这方面，使用商业电极是可取的，但它们的获取可能既复杂又昂贵。手术是该协议中最关键的步骤之一。强烈建议甚至有必要聘请经验丰富的外科医生，特别是对于电极植入。由于手术经常需要延长麻醉时间，有时还需要在手术过程中进行焊接应用，因此每个实验室必须对每种啮齿动物进行必要的测试，并采用适当的麻醉（可以使用不同的鸡尾酒），特别是在动物饲养条件下，窝之间的差异，甚至动物之间的个体差异。适当的计划和考虑可以防止在手术过程中丢失动物。电极植入是另一个关键步骤。它需要非常小心，以避免打头骨并损坏脑膜或脑组织。螺钉应正确放置，即完全固定在颅骨中，否则信号上会出现噪声和伪影，例如与不使用脑电图记录的糟糕的托管或运动相关的信号。必须始终进行和观察术前和术后治疗和条件，以避免啮齿动物的痛苦。皮下注射利多卡因可用于头部皮肤，然后再用手术刀切开。向动物的眼睛滴一滴盐水将有助于防止干燥。此外，必须在口腔中给予盐水溶液，手术后，必须皮下或腹膜内给予1mL，以补偿动物的体液平衡并防止脱水。手术后，必须立即在牙水泥帽所在的头皮周围使用抗炎药物（减轻疼痛）以及通过皮下或局部抗生素使用的抗生素（以减少感染的可能性）。手术后24小时重复上述程序。脑电放大器在动物背部的位置是同时记录的主要困难。背心的设计和制造专门基于动物的大小。背心必须允许啮齿动物的自然运动（见 图5）。后者将保证该技术的主要优势，即记录自由运动。由于动物在手术后和随后的几天内没有试图移除背心、头部连接器或电缆，因此推测该装置没有显着产生运动限制或引起疼痛或不适。为了根据BTS标记的事件在时期进行正确的EEG分割，必须写下定义明确的协议。临时标记可以通过时间序列操作合并，因为两个系统使用相同的时钟来设置其时间戳。以上扩展了结合电生理数据进行分析的动物实验的可能性。
这里介绍的技术可用于任何神经科学研究领域，以及最常见的鼠种甚至其他物种。行为跟踪软件的多功能性是最重要的优势之一，因为它可用于迷宫的多功能性，如莫里斯水迷宫、开阔场地、新型物体识别、条件位置偏好、孔板、高架加迷宫、Y 迷宫、径臂迷宫、巴恩斯迷宫等。它最多可同时使用16台摄像机。此外，可以报告数百种不同的措施（有关更多详细信息，请参阅手册31，32）。考虑到这项工作描述了脑电图记录的实验，其他一些技术，如局部场电位或单单元记录是可能的。但是，用户必须考虑到常规设置和几个准备步骤需要更改以用于其他目的。因此，当这种技术与EEG Wi-Fi记录一起使用时，可能性得到了扩展，因为它为动物研究增加了新的视角，例如对人类进行的研究，以评估EEG整合和动力学的几个特征，如连接性，EEG波段功率或诱发反应。与人类不同，动物实验可以评估药物管理，基因修饰或表达，以及许多其他实验范式。对于脑电图分析，考虑到某些协议对所需行为的重复次数非常少，这限制了平均反应并获得可靠结果的可能性。因此，在开始实验之前，请仔细设计被认为要执行的记录和分析方案。然而，必须考虑到，在动物实验中工作不可能阻止运动，这增加了实验方案的复杂性以及信号分析和行为任务的考虑。目前，用于完整跟踪系统和脑电图记录的设备不是标准化或模块化的，这意味着它们的设置旨在单一协议并适应探索其他行为任务，这意味着/暗示大量实验室的成本更高。这种情况可以通过遵循本研究中解释的选项来解决。然而，对于更可靠的实验，可以实现一些改进。这项工作可以通过几个步骤进行改进，从电极制造到行为和信号处理。尽管如此，事实证明，使用负担得起的高科技但廉价的设置可以进行动物跟踪和脑电图采集。
总之，目前的工作试图帮助科学家，特别是在神经科学领域，能够结合使用这两种不常用的技术。使用行为跟踪软件的脑电图和行为测试的同时记录技术具有许多优点，并且在神经科学的许多领域特别有用，特别是在学习和记忆领域。考虑到该设备具有其他功能，可以作为海马体的皮层下结构的深度记录，但如前所述，几个准备步骤将发生变化。无线设备几乎解决了传统电线方法的所有限制，例如动物从一个笼子到另一个笼子的移动问题，阻碍或缠绕动物与电缆。如上所述，这种设置技术是用户友好的，几乎未经培训或非专业的专家或个人小组可以使用该软件。脑电图设备的价格低于普通脑电放大器。行为跟踪软件也是市场上最实惠的视频跟踪软件之一。该软件需要年度许可证。该设备可用于多个实验装置、不同的动物和多功能类型。我们希望这项工作将有助于科学界，并提供同时研究行为和脑电图的便捷途径。

行为测试在神经科学研究中对于在体内环境中产生的大量信息至关重要。在这方面，研究人员已经广泛使用不同的行为测试来分析感觉运动功能、社交互动、焦虑样和抑郁样行为、物质依赖和各种形式的认知功能1。即使对于大多数专家观察者来说，手动记录行为测试也可能很困难、令人筋疲力尽且不准确。尽管已经做出了一些努力来开发用于行为登记的免费和开源软件（例如，用于性行为的 sexrat male2 应用程序），但有几种替代方案允许自动和实时记录从鱼3 到啮齿动物4，5，6 的不同动物物种的行为。视频跟踪是一种有价值的方法，用于快速准确的行为记录，用于各种应用7.行为记录区的一个更潜在的特征是探索行为表现过程中的神经活动。同时记录神经元活动（从单个细胞到主要大脑区域）和行为任务可以向我们展示大脑如何产生特定的行为模式8。行为是一系列次要成分，可以揭示神经活动与运动或动作之间的相关性。如果可以通过多个时间尺度同时记录神经元活动和行为模式，他们可以解释每种大脑状态如何与每种特定行为相关联（有关行为记录的更深入检查，请参阅Datta等人，2019 年评论8）。因此，以所需规模（从神经元到大脑的大面积）同步记录行为和神经元活动被认为是一种非常有用的工具。有几种系统旨在将行为记录与其他测量值集成为神经活动4，5。
尽管脑电图被认为是临床和研究神经科学领域使用最广泛的技术之一，但相对较高的移动性以及脑电图记录装置的尺寸使该技术在体内模型9的情况下检测独特且具有挑战性。已经开发出一些解决这个问题的方法，例如，使用电缆和旋转装置，允许动物在竞技场中自由移动。然而，基于电缆的系统经常给进行研究带来问题，例如，在将动物从一个笼子转移到另一个笼子的过程中，观察到动物与电缆的阻碍或缠绕。已经开发了用于无线电生理记录的遥测设备，以增加记录情况的灵活性10，11。然而，由于记录通道数量少和采样率低，此类系统显示出相当大的局限性11。在这项研究中，我们使用了一种商用无线系统，该系统通过Wi-Fi连接与自由移动的啮齿动物系统12发送来自动物的脑电图信号。该设备重 6 克，最多可容纳 16 个通道，以 1 kSps 的速度录制。该系统允许在动物环境中进行脑电图或尖峰记录，减少干扰，与市场上的传统电生理系统相比，这是一种经济的解决方案。此外，我们还使用视频跟踪软件同步了这些数据，以提供脑电图和行为模式之间的相关性。这种同步是通过根据两个系统生成的时间戳对齐和插值数据和事件来离线完成的，并在 MATLAB 上进行处理。
成体神经发生被定义为动物齿状回中新生成的细胞在神经元中的增殖、存活和分化13，14。已知该过程与记忆和学习改善有关，通过富集环境（EE）条件增加啮齿动物的成年神经发生15。EE包括将啮齿动物成群结队地安置在配有玩具和管子的大笼子内，其中动物具有新颖而复杂的动物，但没有生物学相关性15。虽然EE刺激海马神经发生，但它在许多因素上也有所不同，例如年龄，动物品系，特定的刺激条件或神经发生检测程序。在暴露于EE外壳7天的中年小鼠中，据报道在海马齿状回（DG）中诞生了新的颗粒细胞（GC）16。试图选择性消融成年大鼠成体神经发生的研究表明，在学习反应中需要约1-2周龄的新颗粒细胞17。在GC在成人DG出生后约2或3周，开始出现一些特征，例如树突棘，这对于兴奋性突触传递18至关重要。Zhao等人进行了定量分析，表明脊柱生长的高峰出现在前3-4周19。几项电生理体内研究表明，仅三周的EE外壳条件就会改变DG的突触传递并增加细胞兴奋性20。此外，据报道，在注射BrdU后1-4周暴露于富集环境中显着增加了小鼠DG颗粒层中BrdU / NeuN细胞的密度21。这些作者认为，由于观察到新神经元数量的大幅增加，因此在EE暴露后一到三周之间存在关键期21。由于没有直接证据，对人类成人海马神经发生（AHN）的研究一直存在争议。然而，最近的一份报告描述了AHN在人类成人大脑中的发育阶段，在DG中识别了数千个未成熟的神经元，从而证明了AHN在人类衰老过程中的重要性22。基于前面提到的证据，在动物模型中研究AHN比以往任何时候都更加重要（有关AHN的更深入检查，请参阅Leal-Galicia等人，2019年综述15）。
如前所述，海马体与学习和记忆能力的基本功能有关。记忆的形成经历了三个不同的过程:编码（记忆获取）、巩固（记忆存储）和检索（记忆识别）23。使用视觉配对比较任务24测试人类的识别记忆。人类和动物记忆和健忘症模型的基础是行为测试，评估识别先前呈现的刺激的能力25，26，就像视觉配对比较任务在人类中所做的那样。因此，用于评估啮齿动物识别先前呈现的刺激的能力（即学习和记忆能力）最常用的行为测试之一是自发的新物体识别任务（NORT）23，27。在采集试验中，NORT协议由两个相同的新物体组成，在熟悉的竞技场中持续10分钟。在0 28和48小时29之间的特定时间（根据每个协议的不同时间）之后，动物被送回包含相同熟悉物体之一和一个新物体的同一竞技场。如果记住了熟悉的对象，动物就会自发地探索新对象26.偏好比率通常用于评估勘探绩效。它是通过将总物体探索时间与新奇或熟悉物体的探索时间相除来确定的。与其他识别记忆测试相比，NORT具有一些优势。最重要的是，它不需要外部动机、奖励或惩罚。它不会产生压力条件。最后，不需要训练来唤起探索对象的行为（有关NORT的更深入检查，请参阅ref.23）。
因此，同时记录多种数据模式及其在学习和记忆研究中的整合，作为成人海马神经发生的影响是非常有吸引力的，并为该领域的研究人员提供了一个令人信服的解决方案。本工作将揭示同步行为视频跟踪评估（新型物体识别任务）和无线脑电图记录所涉及的所有过程。在这里，我们回顾了电极制造工艺，硬膜外（颅骨螺钉）电极植入手术，环境富集方案（用于海马神经发生诱导），遵循NORT协议，BTS设置，用于实时同时监测的EEG – BTS耦合，以及在MATLAB计算环境中执行的脑电图和行为数据分析。

<table><tbody><tr><td>#2 Variable speed rotary tool tip</td><td>Reorder #310048, Lenght 44.5mm</td><td>SS White</td><td>For making the holes where the screws will be inserted</td></tr><tr><td>#4 Scalpel and blade</td><td></td><td></td><td></td></tr><tr><td>50 X 50 X 50 cm Open Field Black Mate Arena</td><td></td><td></td><td></td></tr><tr><td>8 pin Receptacle Housing Female</td><td>Amphenol FCI</td><td>10147606-00008LF</td><td></td></tr><tr><td>8 pin Receptacle Housing Male</td><td>Amphenol FCI</td><td>10147603-00008LF</td><td></td></tr><tr><td>Acrylic Resin</td><td>MDC Dental</td><td>NicTone</td><td>For fixating the screws to the skull</td></tr><tr><td>ANY-maze video tracking software</td><td>Stoelting, Co.</td><td>version 6.1</td><td>&lt;u&gt;http://www.anymaze.co.uk/)&lt;/u&gt;</td></tr><tr><td>benzalkonium chloride antiseptic solution</td><td>Benzal</td><td>Benzal</td><td></td></tr><tr><td>Bulldog clamps</td><td>Cientifica VelaQuin</td><td></td><td>For retracting the skin</td></tr><tr><td>Camera</td><td>Logitech</td><td>c920</td><td></td></tr><tr><td>Copper wire</td><td></td><td></td><td></td></tr><tr><td>Crimp contact</td><td>Amphenol FCI</td><td>10147604-01LF</td><td></td></tr><tr><td>DELL PC</td><td>DELL</td><td></td><td></td></tr><tr><td>Electrode</td><td></td><td></td><td></td></tr><tr><td>JAGA16</td><td>Jinga-Hi, Inc.</td><td>JAGA16</td><td></td></tr><tr><td>Ketamine</td><td>PiSA Agropecuaria</td><td>ANESKET</td><td>For anesthesia</td></tr><tr><td>MATLAB</td><td>R2020a</td><td>MathWorks</td><td>Script was develop ped in collaboration with Jinga-Hi, Inc.</td></tr><tr><td>Monomer</td><td>MDC Dental</td><td>NicTone</td><td>For fixating the screws to the skull</td></tr><tr><td>Neurophys software</td><td>Jinga-Hi, Inc./ Neurosys, LLC</td><td>Neurosys 3.0.0.7</td><td></td></tr><tr><td>Screwdrive</td><td></td><td></td><td>For inserting the screws into the skull</td></tr><tr><td>Screws</td><td></td><td></td><td></td></tr><tr><td>Screws equiped with electrode</td><td></td><td></td><td></td></tr><tr><td>Stereotaxic instrument</td><td>KOPF</td><td></td><td>For the surgery</td></tr><tr><td>Variable speed rotary tool</td><td>Dremel 3000</td><td>Dremel</td><td>For making the holes where the screws will be inserted</td></tr><tr><td>Voltmeter</td><td>PROAM</td><td>MUL-040</td><td>For confirming that the electrode conducts electricity</td></tr><tr><td>Xilazine</td><td>PiSA Agropecuaria</td><td>PROCIN</td><td>For anesthesia</td></tr></tbody></table>

simultaneous monitoring of wireless electrophysiology and memory behavioral test as a tool to study hippocampal neurogenesis

从脑电图（EEG）获得的脑电波振幅已被公认为动物和人类认知能力，记忆和学习的基础。成人神经发生机制也与记忆和学习改善有关。传统上，研究人员过去常常通过行为任务来评估啮齿动物模型中的学习和记忆参数。因此，同时监测行为变化和脑电图在关联大脑活动和任务相关行为之间的数据方面特别有趣。然而，进行这两项研究所需的大多数设备要么复杂，昂贵，要么使用阻碍自然动物运动的有线设置网络。在这项研究中，用无线电生理学设备记录了脑电图，同时执行了一种新的物体识别任务（NORT）。动物的行为由视频跟踪系统同时监控。两个记录都通过它们的时间戳进行离线分析，这些时间戳同步以将脑电图信号与动物的动作联系起来。受试者包括中期环境富集处理后的成年Wistar大鼠。六个颅骨螺钉电极成对固定在两个半球的额叶、中央和顶叶区域，并参考位于鼻骨后方的电极。NORT协议包括将动物暴露于两个相同的物体10分钟。2小时和24小时后，其中一个物体被一个新的物体替换。每个物体的探索时间由行为跟踪软件（BTS）和脑电图数据记录监测。与行为数据同步的脑电图分析包括对α和β相对带功率的估计，以及在三个实验阶段之间新颖对象识别与熟悉对象探索之间的比较。在本稿中，我们讨论了电极制造工艺、硬膜外电极植入手术、环境富集方案、NORT 方案、BTS 设置、用于实时同时监测的 EEG-BTS 耦合以及基于自动事件检测的 EEG 数据分析。

应用上述方法在环境富集处理后同时记录脑电图和大鼠活性。三个月大的雄性Wistar大鼠接受中期环境富集治疗方案20天，并操作它们将六个颅骨螺旋电极固定在额叶，中央和顶叶区域，参考位于新西兰的第七个电极。动物在自然的暗光条件下饲养，可以 随意 获得食物和水。这项工作显示了EEG系统和行为跟踪软件之间的集成，用于同时进行现场记录。我们只使用在EE方案下治疗的动物，因为我们不假装比较治疗的有效性，而只是举例说明设备的优点。作为使用的20天环境富集住房方案刺激成人神经发生的证据，我们从我们实验室未发表的数据中提供了来自EE下动物和在标准条件下饲养的动物的BrdU阳性细胞计数数据。使用三个月大的雄性Wistar大鼠。他们用BrdU注射三次，彼此之间间隔12小时。麻醉动物（戊巴比妥（50mg / kg，ip）并通过经心灌注安乐死（见 图5）。为了确保附着在脑电图装置上的背心不会限制动物的运动，我们将开放现场测试（OFT）分为两组，一组在佩戴设备（背心和脑电放大器）时接受手术，另一组动物在没有佩戴硬件的情况下保持完整。在10分钟的测试中，我们没有发现动物行进的距离有显着差异（见 图5）。典型的 NORT 协议包括呈现两个对象，以及用新对象替换其中一个对象。行为跟踪软件监控探索时间。
行为跟踪软件记录了一组动物，以评估它们的关键性能参数。因此，我们使用三个参数来评估勘探性能。偏好比率是使用动物头部在物体区域花费的时间计算的，该时间报告了动物头部在每个物体上花费的总时间。此外，我们计算了向物体移动所花费的时间的偏好比率，该比率显示了向每个物体区域移动的每只动物所花费的总时间。此外，还计算了每次访问每个对象所花费的时间。 图 6 显示了上述三个参数结果。在获取试验中，三个评估参数中的物体之间没有区别:三项试验在物体区域的头部时间，三项试验向物体移动的时间以及每个物体的每次访问时间。STM试验没有差异。同时，在LTM试验中，新对象的探索首选比率显着更高。此外，在LTM试验中，也可以看到每次访问所花费的时间（图C）对新对象的偏好。视频 1 显示了实验中记录的大鼠的代表性示例，而 视频2 显示了同时进行脑电图和行为记录的代表性示例。
可以使用计算机的时钟将跟踪的时间事件与行为跟踪和脑电图软件记录相匹配。图7和图8显示了脑电图相对功率在α和β带上的变化。这些与运动控制、注意力和记忆有关，这表明探索只与这些功能有关。动物3的结果表明，关于ACQ和LTM的STM的α功率趋于降低，这表明与探索或记忆检索相关的不同步。对象识别（处理时期）的数量很少。到目前为止，无法确定统计检验是否会验证这种差异是否真实，或者工件是否能够产生这样的实验条件。然而，通过同时标记动物事件和为未来研究项目产生的脑电图结果的时间表，时代分割、标记和分析已经成为可能。结合这些系统可以防止通过手动标记过程错误地识别事件，这已成为动物实验目的中的一个重大问题。BTS和电生理（EP）活性的结合可以准确地与动物行为相关联;然而，实验条件需要使用先进的信号处理技术来消除运动伪影并有效地改进实验设置。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61494/61494fig01.jpg"> 图 1:富集环境 （EE） 条件笼的示例。 住房里提供了玩具和管子，动物在其中发现新奇和复杂，但没有生物学相关性。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig01large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61494/61494fig02.jpg"> 图2:硬膜外电极在大鼠头骨中的位置。 螺钉同时用作耳机的锚和电极。F = 正面;C = 额顶;P = 顶叶;3 = 左;4 = 右;NZ = 作为地面参考。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig02large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61494/61494fig03.jpg"> 图3:硬膜外（颅骨螺钉）电极植入手术的代表性图像。 图像显示大鼠在手术不同阶段植入的颅内电极螺钉。确保在执行此过程时遵循无菌技术。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig03large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61494/61494fig04.jpg"> 图4:大鼠的代表性图像以及实验装置。 大鼠被要求在用于NORT协议的竞技场内穿上带有嵌入式电池的脑电图装置上的背心。该图显示了安装在头部老鼠上的耳机和电缆连接器。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig04large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61494/61494fig05.jpg"> 图 5:运动能力和 EE 方案的成人神经发生刺激的证据。 （A）露天测试（OFT）中动物活动10分钟的代表性图像，以及佩戴设备/手术的动物和没有设备/无手术的动物的平均距离。（乙-E）具有 BrdU 标记细胞（深色）的代表性 DG 切片，用于 EE 和标准外壳组。图B和D显示DG的低放大倍率，图C和E显示较高放大倍率下的盒子区域。图B和C是来自EE外壳组的组织，面板D和E来自标准外壳组。插图说明了两组中标记单元格的平均数量。ML - 分子层;GCL – 颗粒细胞层;SGZ – 亚颗粒区;箭头 - BrdU+ 细胞。图表显示了 SEM ±平均值。T-学生测试用于比较组。* 第≤0.05页。在开放现场测试中，各组之间未发现显著差异。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig05large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61494/61494fig06.jpg"> 图6:NORT评估中的勘探绩效。 （A）三次试验在目标区的头部时间。（B）向三个试验对象移动的时间。（C） 每个对象每次访问的时间。图表显示了 SEM ±平均值。 在所有参数中使用了双向重复测量方差分析与Sidak多重比较检验。* p≤0.05， ** p≤0.01 在各自试验的对象之间。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig06large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61494/61494fig07.jpg"> 图 7:与探索相关的 alpha EEG 波段功率的变化。 这张图显示了动物开始探索物体后相对阿尔法功率的变化，从半秒到2.5秒。这六张图分别对应于额电极、中央电极和顶电极（从上到下）以及左右两侧。箱线图显示了对象（"熟悉"和"新"）以及阶段:"ACQ"、"STM"和"LTM"的每个条件组合的此类时间序列的分布。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig07large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61494/61494fig08.jpg"> 图 8:与探索相关的 β EEG 带功率的变化。 该图显示了动物开始探索物体后相对β功率的变化，从半秒到2.5秒。这六张图分别对应于额电极、中央电极和顶电极（从上到下）以及左右两侧。箱线图显示了对象（"熟悉"和"新"）以及阶段:"ACQ"、"STM"和"LTM"的每个条件组合的此类时间序列的分布。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61494/61494fig08large.jpg" target="_blank">请点击此处查看此图的大图。</a>
视频1:显示实验中记录的大鼠的代表性视频。大鼠在用于NORT协议的竞技场内。大鼠穿着带有嵌入式电池的背心连接到脑电图装置。<a href="https://cloudflare.jove.com/files/ftp_upload/61494/VIDEO_1.mp4" target="_blank">请点击此处下载此视频。</a>
视频2:代表性视频，同时显示脑电图和行为记录。脑电图信号显示在左侧，行为测试（NORT）显示在视频的右侧。<a href="https://cloudflare.jove.com/files/ftp_upload/61494/VIDEO_2.mp4" target="_blank">请点击此处下载此视频。</a>

Watch this Scientific Journal Video about Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis at JoVE.com

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

此处介绍的协议提供了有关同步脑电图（EEG）和实时行为评估的信息。我们已经讨论了该协议中涉及的所有步骤，对于神经科学的许多领域的研究人员来说，这是一个有吸引力的解决方案，特别是在学习和记忆领域。

同时监测无线电生理学和记忆行为测试作为研究海马神经发生的工具

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

simultaneous-monitoring-wireless-electrophysiology-memory-behavioral

Universidad de Cartagena UDEC

Research

JoVE Journal

Behavior

2.8K 次观看.  Universidad Iberoamericana Ciudad de México. 此处介绍的协议提供了有关同步脑电图（EEG）和实时行为评估的信息。我们已经讨论了该协议中涉及的所有步骤，对于神经科学的许多领域的研究人员来说，这是一个有吸引力的解决方案，特别是在学习和记忆领域。

视频: Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

科研

遗传学

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

同时脑电图，实时测量的啮齿动物的大脑皮质中的神经元活动的乳酸浓度与Optogenetic的操作

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

神经科学

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行为学

Long-term Continuous EEG Monitoring in Small Rodent Models of Human Disease Using the Epoch Wireless Transmitter System

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test interventions at various stages of the disease process. These animal models are particularly difficult to implement in immature rodents, a classic model organism for laboratory study of these disorders. Recording continuous EEG in young animal models of seizures and other neurological disorders presents a technical challenge due to the small physical size of young rodents and their dependence on the dam prior to weaning. Therefore, there is not only a clear need for improving pre-clinical research that will better identify those therapies suitable for translation to the clinic but also a need for new devices capable of recording continuous EEG in immature rodents. Here, we describe the technology behind and demonstrate the use of a novel miniature telemetry system, specifically engineered for use in immature rats or mice, which is also effective for use in adult animals.

Here we demonstrate the use of a wireless enabling technology for electroencephalogram (EEG) in neonatal rodent models of human disease. With telemetry, there are no encumbering connections, thus allowing natural behaviors.

在人类疾病中使用大纪元无线传输系统的小型啮齿动物模型的长期连续监测脑电图

Many progressive neurologic diseases in humans, such as epilepsy, require pre-clinical animal models that slowly develop the disease in order to test ...

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

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced electrodes for large-scale electrophysiological recording of neuronal activity, but the procedures for their chronic implantation are still underdeveloped. The activity of hippocampal place cells is known to correlate with an animal&#39;s position in the environment, but the underlying mechanisms are still unclear. To investigate place cells, here we describe a set of techniques which range from the fabrication of devices for chronic silicon probe implants to the monitoring of place field activity in a cue-enriched treadmill apparatus. A micro-drive and a hat are built by fitting and fastening together 3D-printed plastic parts. A silicon probe is mounted on the micro-drive, cleaned, and coated with dye. A first surgery is performed to fix the hat on the skull of a mouse. Small landmarks are fabricated and attached to the belt of a treadmill. The mouse is trained to run head-fixed on the treadmill. A second surgery is performed to implant the silicon probe in the hippocampus, following which broadband electrophysiological signals are recorded. Finally, the silicon probe is recovered and cleaned for reuse. The analysis of place cell activity in the treadmill reveals a diversity of place field mechanisms, outlining the benefit of the approach.

We describe the diverse steps to implant chronic silicon probes and to record place cells in mice that are running head-fixed on a cue-enriched treadmill apparatus.

富氧运动器械中慢性硅探针的植入及海马位细胞的记录

An important requisite for understanding brain function is the identification of behavior and cell activity correlates. Silicon probes are advanced ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

啮齿动物联合局部脑电图和局部电位的同步记录

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Recording Spatially Restricted Oscillations in the Hippocampus of Behaving Mice

The local field potential (LFP) emerges from ion movements across neural membranes. Since the voltage recorded by LFP electrodes reflects the summed electrical field of a large volume of brain tissue, extracting information about local activity is challenging. Studying neuronal microcircuits, however, requires a reliable distinction between truly local events and volume-conducted signals originating in distant brain areas. Current source density (CSD) analysis offers a solution for this problem by providing information about current sinks and sources in the vicinity of the electrodes. In brain areas with laminar cytoarchitecture such as the hippocampus, one-dimensional CSD can be obtained by estimating the second spatial derivative of the LFP. Here, we describe a method to record multilaminar LFPs using linear silicon probes implanted into the dorsal hippocampus. CSD traces are computed along individual shanks of the probe. This protocol thus describes a procedure to resolve spatially restricted neuronal network oscillations in the hippocampus of freely moving mice.

This protocol describes the recording of local field potentials with multi-shank linear silicon probes. Conversion of the signals using current source density analysis allows the reconstruction of local electrical activity in the mouse hippocampus. With this technique, spatially restricted brain oscillations can be studied in freely moving mice.

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 ...

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 ...