这项工作得到了京都大学白尾项目、挑战研究补助金（先锋）（No.22K18644）、科学研究补助金 （C） （No. 22K12745）、科学研究补助金 （B） （No. 21H02851） 和科学研究补助金 （A） （No. 19H01039） 的支持。感谢 意得辑 （www.editage.jp） 的英文编辑工作。

所有实验均经 EHUB 动物实验委员会（No.2022-003、2023-104）批准，并按照 EHUB 发布的《实验室灵长类动物护理和使用指南》进行。实验使用了 9 只普通狨猴 （Callithrix jacchus，6 只雄性和 3 只雌性，2-12 岁，体重 330-490 g）。
1. 动物
<ol><li>将狨猴安置在配备巢箱、木制栖木和其他丰富装置的单个笼子中。</li>
	<li>将房间保持在 12 小时的光暗循环下，温度和湿度分别保持在 28 ± 2 °C 和 40 ± 20%。</li>
	<li>每天两次给动物喂食 14 克新世界猴子颗粒，并辅以阿拉伯树胶和黄粉虫等食物。 随意提供水。</li>
	<li>在实验室的消音箱中进行所有实验。</li>
</ol>2. 设备（图 1B 和材料表）
<ol><li>使用 4 mm 银电极。电极表面的 AgCl 涂层可防止极化并确保稳定记录。</li>
	<li>使用放大器记录脑电图信号。带通滤波器 （0.016-250 Hz） 并以 1,000 Hz 对数据进行采样。</li>
	<li>将 64 通道电极输入盒连接到放大器，将其放置在被摄体前面。</li>
	<li>将扬声器放置在距离狨猴头部 30 厘米处，并将声级控制在 65-75 分贝，如在耳朵位置测量的那样。通过此扬声器传递听觉刺激。</li>
	<li>将摄像头放在拍摄对象前面，以在脑电图记录期间监测他们的状况。</li>
	<li>灵长类动物椅子由亚克力板和合成树脂柱制成。对于实验，让研究人员抱着坐在脚踏板上的动物;此时，插入颈件并将其固定到颈板上，然后将腰件插入并固定到腰板上。确保每只狨猴的整个身体都松散地固定。</li>
</ol>3. 麻醉
<ol><li>通过肌肉注射阿法沙酮 （6-8 mg/kg） 和阿托品 （0.05 mg/kg） 麻醉动物。该方案允许麻醉维持约 20 分钟。如果手术时间较长，则给予额外剂量的阿法沙隆以延长麻醉持续时间。还要事先给予止吐药（Maropitant 1 mg/kg，皮下注射）以对抗恶心，这是阿法沙酮的副作用。 注意：狨猴在手术过程中必须接受注射或吸入麻醉，并且应在剃毛后立即恢复。</li>
	<li>使用脉搏血氧仪监测生命体征，必要时给予氧气-空气混合物（O2 0.5 L/min，空气 0.5 L/min）。</li>
	<li>将室温保持在 27 °C 以上，并在狨猴的身体周围包裹一块温暖的布，以防止体温过低。</li>
</ol>4. 脱毛
<ol><li>用电动剃须刀剃光整个头部（包括耳廓后面）。</li>
	<li>为敏感皮肤涂抹脱毛霜。5 分钟后用湿纱布擦去乳霜。</li>
</ol>5. 掩模准备
<ol><li>提前加工热塑性口罩以适合猴椅的尺寸。具体来说，在从椅子的垂直长度突出的靠背部分钻上四个螺丝孔，使其可以固定在椅子的颈板上。 注意：热塑性面罩是安全的，可用于在患者放射治疗期间固定头部。我们使用了一个专为儿童设计的小面具，并将其剪裁成适合狨猴的大小。这种面罩在 75 °C 的温水中时会变软，从水中取出时会随着温度的降低而变硬。</li>
	<li>对于实验，将动物放在灵长类动物的椅子上。使用颈板和腰板支撑麻醉的动物，并将动物和这些板固定在椅子上。</li>
	<li>在热水中加热面膜，然后将其成型以适合狨猴的头部。从温水中取出口罩后，等待温度下降（大约在 50s °C 范围内）以防止烫伤，然后将其放在受试者的头上以塑造口罩。</li>
	<li>冷却和硬化后，从动物身上取下面罩，切出面罩的头部和耳朵部分，露出电极设置的区域。 注意：一旦制作了这个面具，它就可以用于其他人。</li>
</ol>6. 椅子和面罩适应（30 分钟/天，持续 3 天）
<ol><li>在清醒的情况下，将动物放在椅子上并奖励它大约 30 分钟，使它适应于椅子上。重复此过程 3 天。</li>
	<li>使用面罩使动物习惯于头部固定 2 天。 注意：这个适应过程是根据个人的情况定制的。适应后，动物在捕捉和戴头罩时可能会暂时抵抗，但一旦它们坐在椅子上并固定头部，它们就会变得平静。</li>
	<li>在椅子适应期间，评估以下行为参数：i） 发出焦虑或警报的声音，ii） 拒绝提供的奖励，以及 iii） 剧烈运动。如果观察到这些行为中的任何一个，请终止当天的会话，让狨猴逐渐适应实验环境。</li>
</ol>7. 脑电图记录（2 小时/天）
<ol><li>科目准备<ol><li>将转运笼连接到家笼的小窗户上，通常自己将狨猴从家笼移动到转运笼。用布盖住携带笼，将其转移到实验室。</li>
		<li>头部固定<ol><li>使用防护手套捕捉狨猴并将其放在特殊的椅子上。</li>
			<li>将准备好的面膜放在狨猴的头上。将连接到椅子上的螺钉体穿过面罩上的孔，并使用翼形螺钉固定。</li>
			<li>用鼻孔下方的带子轻轻固定头部和面罩。 注意：只要电极不因移动而移开，就不需要完全固定受试者的头部。因此，我们在所有受试者中使用相同的掩码。</li>
		</ol></li>
		<li>电极位置的定义<ol><li>使用鼻、鼻和耳垂作为解剖标志，根据国际 10-20 方法32、33、34、35、36、37 确定电极的位置。用卷尺测量中线 nasion 和 inion 之间的距离。定义 Cz 在距离中间的位置。将其他电极（Fz、Pz、Oz、F3 或 F4）定位为离子-离子长度的 20% 作为电极间距。使用油性皮肤图标记头皮上的电极位置。</li>
		</ol></li>
		<li>皮肤准备<ol><li>用蘸有外用酒精的细棉签擦拭标记区域，以去除头皮上的污垢和皮脂。</li>
		</ol></li>
	</ol></li>
	<li>电极设置 注：在所有电极位置执行步骤 7.2.1-7.2.3，将电极放置在 Fz、Cz、C3、C4、Pz、A1、A2 和 F3（或 F4）处。<ol><li>使用切管器，将一根内径为 4 mm、外径为 7 mm 的硅胶管切割成约 20 mm 的长度。</li>
		<li>将粘合剂涂在切割管的边缘并将其粘附在头皮上。</li>
		<li>使用注射器和非尖头注射器针头用 EEG 凝胶填充管内。</li>
		<li>将参比电极连接到 Pz，将接地电极连接到 F3 或 F4（图 1C）。</li>
		<li>将电极插入管中，并将电极电缆连接到输入框。</li>
		<li>启动 EEG 记录应用程序以测量电极阻抗，并调整参数以确保所有电极均低于 5 kΩ。</li>
		<li>捆绑电极电缆以降低噪音。</li>
	</ol></li>
	<li>通过使用注射器手动施用 1-3 mL 液体奖励（口香糖或营养品），在头部固定程序期间和任务会话之间给予奖励。</li>
	<li>指定要保存在 EEG 记录软件上的文件，然后按 Start Recording 按钮 。在 EEG 记录开始后立即运行刺激呈现脚本。刺激呈现脚本执行完成后，按下 EEG 记录上的 停止 按钮以结束所有记录。录制完成后，取下电极和头罩。</li>
	<li>捕捉动物并将它们放回它们的笼子里。将管子留在头皮上;所有管子在大约 1 天内自然脱落。 注意：在早期，我们使用丙酮溶解胶粘剂，并在录音后取出管子;然而，也有皮肤损伤的案例;因此，我们随后将动物放回了它们的家笼中，没有取下管子。在我们迄今为止进行的数十次实验中，狨猴对取出的试管没有表现出任何兴趣，也从未发生过意外摄入。</li>
	<li>刺激物<ol><li>在圈养室或实验室中记录狨猴的自然、简单和复合呼叫，这些呼叫在后续的脑电图记录中未使用。从录制的文件中提取三种物种特异性的简单和复合调用（Phee、Tsik-Ek 和 Tsik-String5 调用）。</li>
		<li>除了呼叫刺激之外，使用编程软件中的函数创建白噪声并将其用作刺激。</li>
		<li>要遵循此协议，请在实验中使用三个 marmoset 调用的听觉文件（16 位，48 kHz）。使用自定义脚本控制任务。 注意：每个区块包含每个刺激的 50 个调用，总共 200 个调用。每个记录块将持续大约 10 分钟。每个参与者必须执行两个模块。呼叫间隔为 3 秒。在四个音频刺激文件中（参见 补充文件 1）， Phee 呼叫刺激大约是 2 秒长，而其他三个大约是 1 秒长。这是因为单个 Phee 调用仍然是一个持久的调用（图 1D）。</li>
		<li>在每个声音刺激文件中，右声道包含狨猴叫声或噪声数据，左声道包含刺激开始的触发信号。通过同步设备将此触发信号发送到脑电图记录系统，并将其记录为事件时间。</li>
	</ol></li>
</ol>8. 数据分析
注意：在编程软件和工具箱中编写的原始代码用于对 EEG 数据进行后处理，如下所述（补充文件 2）37。
<ol><li>预处理<ol><li>重新引用链接的耳朵引用。</li>
		<li>2 Hz 的高通滤波器。</li>
		<li>从刺激开始前 100 毫秒到刺激开始后 1000 毫秒的纪元。</li>
		<li>基线校正到 100 毫秒刺激前期的平均值。使用 ±150 μV 的标准拒绝伪影。</li>
	</ol></li>
	<li>绘制事件相关电位 （ERP）<ol><li>每个受试者的所有试验数据的平均值。</li>
		<li>通过对所有受试者求平均值来获得组平均波形。</li>
		<li>要比较呼叫类型和噪声刺激之间的平均 ERP，请应用单因素方差分析 （ANOVA），将刺激作为 Cz 响应中的主体间因子。</li>
		<li>使用 Tukey 方法应用事后多重比较分析。</li>
	</ol></li>
	<li>绘制事件相关频谱扰动 （ERSP）<ol><li>使用公式 （1） 计算 ERSP，以可视化在宽频率范围内随时间变化的频谱功率平均事件相关变化。 Fk （f，t） 是频率 f 和时间 t 处试验 k 的频谱估计值： <img alt="Equation 1" src="/files/ftp_upload/66869/66869eq01.jpg" style="margin: auto;">（1）</li>
		<li>使用正弦小波变换将时频分解应用于活动，在最低频率 （10 Hz） 处具有三个长度周期，在最高频率 （120 Hz） 处，长度随频率线性增加，最高可达 32 个周期。</li>
		<li>定义 2-30 Hz 刺激开始后 150 ms 周期内的初始和瞬态响应，以及 40-100 H 刺激开始后 151 至 950 ms 期间 800 ms 周期内的持续响应。</li>
		<li>要检验受试者年龄和呼叫类型之间 Fz 和 Cz 的初始响应和持续响应的差异，请使用呼叫类型作为主体内因子，使用 年龄 作为主体间因子来执行双向方差分析。</li>
	</ol></li>
</ol>

用于狨猴声音感知比较研究的无创脑电图

作者没有需要披露的利益冲突。

关于麻醉的注意事项 氯胺酮和甲苯噻嗪给药都已被尝试过，虽然它们是镇痛剂，因此适用于长时间的痛苦任务，但狨猴在没有吸氧的情况下往往会出现血氧水平降低44。简而言之，alfaxalon 可能最适合剃须或制作面膜等无痛任务。此外，对于仅需 10-15 分钟的剃须，吸入麻醉将是最合适的。由于异氟醚持续时间短且浓度低 （约 1%），因此在插管期间未使用。
<p cla...

灵长类动物使用物种特异性的发声来传达生物学上重要的信息，例如呼叫者的情绪状态或维持社会纽带的意图、捕食者的存在或其他危险情况。对声音丰富的非人类灵长类动物发声感知背后的神经机制的研究可能为我们提供关键线索，以更好地理解人类语言的进化起源。
普通狨猴是原产于南美洲的小型灵长类动物。近年来，狨猴越来越多地与猕猴一起被用作模式动物，因为它们的繁殖能力高，体型小，易于使用，以及有用的转基因技术的发展 1,2,3。除了作为疾病模型的实用性外，群体内丰富的声音交流是该物种的另一个独特特征 4,5,6,7。狨猴经常交换声音信号，与森林中看不见的同种动物交流。通过检查狨猴发声感知和产生所涉及的大脑活动，我们可以确定它们如何处理大脑中自身或同种呼叫的听觉信息，并确定涉及哪些神经回路。先前的研究表明，初级听觉皮层8、9、10、11、12 和额叶皮层13、14 的神经活动参与狨猴的声音产生。此外，这些兴奋和抑制的神经元反应受到初级听觉皮层中听觉-声音相互作用的调节 8,10。这些研究使用侵入性记录方法提供了单神经元水平的详细神经活动数据。许多研究进一步检查了狨猴发声产生所涉及的神经活动;然而，对声音感知的理解仍然知之甚少15,16。
几项无创脑成像研究阐明了狨猴发声处理的神经机制 17,18,19;它们的高空间分辨率是一个优势，但是，在扫描过程中使动物保持清醒状态需要先进的技术。然而，最近，Jafari 等人使用功能性磁共振成像 （fMRI） 确定了清醒狨猴参与声音感知的额颞区19。几乎所有阐明人类参与发声感知和产生的大脑功能的实验都是使用无创方法进行的，例如头皮脑电图 （EEG）、脑磁图 （MEG）20,21 和 fMRI 22,23,24。许多人类研究调查了使用 EEG 与声音感知相关的大脑活动。这些研究大多集中在情绪信息 25,26,27 和情绪词的显著性 28 上，结果揭示了声音感知过程中事件相关电位的变化29。使用颅内植入电极的人体皮层电图 （ECoG） 和单神经元记录仅在接受神经外科治疗的患者中进行了有限数量的实验30,31。
在了解人类发声感知和产生背后的独特神经机制时，将人类与猴子进行比较的进化观点非常重要。为了直接比较声音丰富的非人类灵长类动物（如狨猴）与人类的语音感知和发声所涉及的神经机制，使用相同的方法比较两个物种之间的数据非常重要。功能性 MRI 允许全脑成像并具有很高的空间分辨率。它的优点是可以记录垂直于颅骨或难以用 EEG 或 MEG 记录的深部区域的活动。然而，MRI 机器的安装和维护成本很高，并且由于设备的性质，可以呈现的刺激存在许多限制。相比之下，EEG、事件相关电位 （ERP） 和 MEG 具有较高的时间分辨率，使其可用于分析时间序列声音处理。特别是，脑电图具有移动性高、能够在各种实验环境中使用、成本相对较低以及只需要一名操作员的优势。
由于已经在人类身上获得了大量的脑电图数据，因此非人类灵长类动物需要使用非侵入性范式的脑电图测量方法。我们的研究小组开发了一种独特的无创脑电图记录方法，使用32 号管对猕猴和狨猴进行记录。在这里，我们报告了有关非人灵长类动物听觉处理的几项新发现 33,34,35,36,37。为了表征响应狨猴物种特异性发声的大脑活动，我们构建了一个实验系统，使用放置在头皮上的电极无创记录大脑活动。在这项研究中，我们描述了狨猴的脑电图测量方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alfaxalone</td>
			<td>Meiji Animal Health</td>
			<td>Alfaxan</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Brain Products</td>
			<td>BrainAmp</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Fuso Pharmaceutical Industries</td>
			<td>Atropine Sulfate Injection</td>
			<td></td>
		</tr>
		<tr>
			<td>Audio editor</td>
			<td>Adobe</td>
			<td>Adobe Audition</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processing software</td>
			<td>MathWorks</td>
			<td>MATLAB</td>
			<td>version R2023a</td>
		</tr>
		<tr>
			<td>Data processing toolbox</td>
			<td>University of California-SanDiego</td>
			<td>EEGLAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processing toolbox</td>
			<td>University of California-Davis</td>
			<td>ERPLAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Panasonic</td>
			<td>ER803PPA</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Unique Medical</td>
			<td>UL-3010</td>
			<td>AgCl coated (custom)</td>
		</tr>
		<tr>
			<td>Electrode gel</td>
			<td>Neurospec AG</td>
			<td>V16 SuperVisc</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode input box</td>
			<td>Brain Products</td>
			<td>EIB64-DUO</td>
			<td>64ch</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>3M</td>
			<td>Scotch 7005S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removering cream</td>
			<td>Kracie</td>
			<td>epilat for sensitive skin</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Bussan Animal Health</td>
			<td>ds isoflurane</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid gum</td>
			<td>San-ei Yakuhin Boeki</td>
			<td>Arabic Call SS</td>
			<td>Gum arabic+water</td>
		</tr>
		<tr>
			<td>Liquid nutrition</td>
			<td>Nestl&#233; Health Science Company</td>
			<td>Isocal 1.0 Junior</td>
			<td>Polymeric formula</td>
		</tr>
		<tr>
			<td>Maropitant</td>
			<td>Zoetis&#160;</td>
			<td>Cerenia injectable solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor Camera</td>
			<td>Intel</td>
			<td>RealSense LiDAR Camera L515</td>
			<td></td>
		</tr>
		<tr>
			<td>Monkey pellets</td>
			<td>Oriental Yeast</td>
			<td>SPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Primate chair</td>
			<td>Natsume Seisakusho</td>
			<td>Order made</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse oximeters</td>
			<td>Covident</td>
			<td>Nellcor</td>
			<td>PM10N</td>
		</tr>
		<tr>
			<td>Skin prepping pasta&#160;</td>
			<td>Mammendorfer Institut f&#252;r Physik und Medizin</td>
			<td>NeuPrep</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicon tube</td>
			<td>AsONE</td>
			<td>&#934;4 x 7mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Fostex</td>
			<td>PM0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Synchronization device</td>
			<td>Brain Vision</td>
			<td>StimTrak</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoplastic mask</td>
			<td>CIVCO</td>
			<td>MTAPU Type Uniframe Thermoplastic Mask 2.4mm</td>
			<td></td>
		</tr>
	</tbody>
</table>

electroencephalography measurements in awake marmosets listening to conspecific vocalizations 

声音交流在灵长类动物的社交互动中起着至关重要的作用，尤其是在生存和社会组织中。人类以语言的形式发展出一种独特而先进的声音交流策略。要研究人类语言的进化，有必要研究人类发声处理背后的神经机制，并通过将它们与非人类灵长类动物的机制进行比较来了解大脑机制是如何进化的。在此，我们开发了一种无创测量清醒非人灵长类动物脑电图 （EEG） 的方法。这种记录方法允许在不伤害动物的情况下进行长期研究，重要的是，它允许我们直接将非人类灵长类动物的脑电图数据与人类数据进行比较，从而深入了解人类语言的进化。在目前的研究中，我们使用头皮脑电图记录方法来研究大脑活动对狨猴物种特异性发声的反应。本研究通过使用头皮脑电图捕捉狨猴在声音感知过程中的广泛神经表征，填补了现有知识的空白，从而提供了新的见解。

首先，我们绘制了狨猴中每种听觉刺激的平均事件相关电位 （ERP）（图 2）。听觉诱发电位 （AEP） 在 噪声 条件下很突出，反映了刺激的清晰开始（见 图 1D）。为了比较呼叫类型和噪声刺激之间的平均 ERP，我们应用了单因素方差分析 （ANOVA），将刺激作为 Cz 响应中的受试者间因子。我们发现刺激分别在刺激开始后 13-18 ms、28-36 ms 和 45-88 ms 对 C...

作者聚焦：使用无创脑电图研究小型灵长类动物的声音信息表示及其精神疾病对声音信息的改变

为了研究语言的进化，比较人类和非人类灵长类动物的大脑机制很重要。我们开发了一种无创测量清醒动物脑电图 （EEG） 的方法。它使我们能够直接长期比较人类和动物之间的脑电图数据，而不会伤害它们。

清醒狨猴的脑电图测量：聆听同种发声 

Vocal communication plays a crucial role in the social interactions of primates, particularly in survival and social organization. Humans have developed a ...

electroencephalography-measurements-awake-marmosets-listening-to

Research

JoVE Journal

Neuroscience

Electroencephalography Measurements in Awake Marmosets Listening to Conspecific Vocalizations 

488 次观看.  Kyoto University. 我们想知道声音信息在富含声音交流的小型灵长类动物的大脑中是如何表示的。我们想说明未来精神疾病（如精神分裂症）将如何改变这种表示。我们比较了四种灵长类动物的非侵入性脑电图，发现人类听觉皮层对声音做出反应的潜伏期比其他灵长类动物长。这将对复杂声音的感知产生重大影响，例如呼叫和语音。很少有实验方法允许直接比较...

视频: 作者聚焦：使用无创脑电图研究小型灵长类动物的声音信息表示及其精神疾病对声音信息的改变

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

调查婴儿和成人使用密集阵脑电图的社会认知（ D脑电图）

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

老年帕金森病大鼠的超声波发声的有针对性的培训

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

记录神经反应和注射示踪剂一个清醒小鼠的制备

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

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 ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

同时脑电图和功能 MRI 期间的可靠脑成像数据获取

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

在麻醉和清醒行为的非人灵长类动物大脑中注射用于光遗传学的AAV载体

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

植入颅窗以在清醒小鼠 中重复体内 成像

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

用于清醒、头部固定大鼠神经成像的头部植入物

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...