这项工作得到了村田科学基金会、铃轩纪念基金会、中谷生物医学工程测量技术促进基金会以及探索性研究（批准号21K19755，日本）和科学研究（B）（批准号23H03416，日本）的资助。

在本研究中，所有动物实验均按照美国国立卫生研究院实验动物护理和使用指南进行，并得到北海道大学机构动物护理和使用委员会的批准。C57BL / 6J小鼠，两只雄性和三只雌性，8至10周龄，用于本研究。这是一个终端过程。这些动物是从商业来源获得的（见 材料表）。
1. 灵活的二维阵列设计和构造
<ol><li>准备所需数量的微型螺旋电极（不锈钢，SUS...

用低成本的 EEG 和毫米级线圈刺激小鼠大脑

<ol>
	<li>Ilmoniemi, R. J., 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=Neuronal+responses+to+magnetic+stimulation+reveal+cortical+reactivity+and+connectivity.">Neuronal responses to magnetic stimulation reveal cortical reactivity and connectivity.</a> Neuroreport. 8 (16), 3537-3540 (1997).</li><li>Hallett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+magnetic+stimulation:+a+primer.">Transcranial magnetic stimulation: a primer.</a> Neuron. 55 (2), 187-199 (2007).</li><li>Thut, G., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+TMS+with+EEG:+How+and+what+for.">Integrating TMS with EEG: How and what for.</a> Brain Topography. 22 (4), 215-218 (2010).</li><li>Ilmoniemi, R. J., Kicic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+combined+TMS+and+EEG.">Methodology for combined TMS and EEG.</a> Brain Topograpy. 22 (4), 233-248 (2010).</li><li>Daskalakis, Z. J., Farzan, F., Radhu, N., Fitzgerald, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+transcranial+magnetic+stimulation+and+electroencephalography:+its+past,+present+and+future.">Combined transcranial magnetic stimulation and electroencephalography: its past, present and future.</a> Brain Research. 1463, 93-107 (2012).</li><li>Tremblay, S., 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=Clinical+utility+and+prospective+of+TMS-EEG.">Clinical utility and prospective of TMS-EEG.</a> Clinical Neurophysiology. 130 (5), 802-844 (2019).</li><li>Pellicciari, M. C., Veniero, D., Miniussi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+cortical+oscillatory+response+to+TMS+pulse.">Characterizing the cortical oscillatory response to TMS pulse.</a> Frontiers in Cellular Neuroscience. 11, 38 (2017).</li><li>Lin, Y. J., Shukla, L., Dugue, L., Valero-Cabre, A., Carrasco, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcranial+magnetic+stimulation+entrains+alpha+oscillatory+activity+in+occipital+cortex.">Transcranial magnetic stimulation entrains alpha oscillatory activity in occipital cortex.</a> Scientific Reports. 11 (1), 18562 (2021).</li><li>Takahashi, S., 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=Laminar+responses+in+the+auditory+cortex+using+a+multielectrode+array+substrate+for+simultaneous+stimulation+and+recording.">Laminar responses in the auditory cortex using a multielectrode array substrate for simultaneous stimulation and recording.</a> IEEJ Transactions Electrical and Electronic Engineering. 14 (2), 303-311 (2019).</li><li>Yoshikawa, T., Higuchi, H., Furukawa, R., Tateno, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+profiles+of+evoked+activity+induced+by+magnetic+stimulation+using+millimeter-sized+coils+in+the+mouse+auditory+cortex+in+vivo.">Temporal and spatial profiles of evoked activity induced by magnetic stimulation using millimeter-sized coils in the mouse auditory cortex in vivo.</a> Brain Research. 1796, 148092 (2022).</li><li>Tang, A. D., 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=Construction+and+evaluation+of+rodent-specific+rTMS+coils.">Construction and evaluation of rodent-specific rTMS coils.</a> Frontiers in Neural Circuits. 10, 47 (2016).</li><li>Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+annealing+and+magnetic+treatment+parameters+to+achieve+high+permeabilities+in+55+Ni-Fe+toroid+cores.">Controlling annealing and magnetic treatment parameters to achieve high permeabilities in 55 Ni-Fe toroid cores.</a> IEEE Transactions on Magnetics. 37 (4), 2315-2317 (2001).</li></ol>

这项研究涉及多点脑电图记录系统与为小动物（包括小鼠）设计的磁刺激系统相结合。构建的系统成本低，易于在生理实验室中构建，并且可以扩展其现有的测量设置。如果这些实验室以前有标准电生理实验的经验，那么从小鼠记录系统获取数据所需的外科手术非常简单。
使用这种方法的一个优点是电极放置在单个动物的头部和头皮上的良好可重复性。用于将螺旋电极分配?...

经颅磁刺激（TMS）因其非/低侵入性而成为人脑科学，临床应用和动物模型研究的有前途的工具。在TMS应用的早期阶段，人类和动物对单脉冲和配对脉冲TMS的皮质效应的测量仅限于运动皮层;易于测量的输出仅限于涉及运动皮层的运动诱发电位和感应肌电位1,2。为了扩展可以通过TMS调制测量的大脑区域，将脑电图（EEG）记录与单脉冲和配对脉冲TMS集成在一起，作为直接检查整个大脑区域的兴奋性，连通性和时空动力学的有用方法3,4,5。因此，同时将TMS和脑电图记录（TMS-EEG）应用于大脑已被用于探测人类和动物的各种浅表皮质大脑区域，以研究皮层内神经回路（见Tremblay等人6）。此外，TMS-EEG系统可用于检查其他皮质时空特征，包括信号传播到其他皮质区域和振荡活动的产生7,8。
然而，由于TMS的非侵入性，TMS在大脑中的作用机制仍然是推测性的，这限制了我们对TMS应用期间大脑功能的知识。因此，从啮齿动物到人类的动物的侵入性转化研究对于了解TMS对神经回路及其活性的影响机制至关重要。特别是，对于动物的TMS-EEG组合实验，尚未为小动物密集开发同步刺激和测量系统。因此，实验者需要根据其特定的实验要求，通过试错来构建这样的系统。此外，小鼠模型在其他 体内 动物物种模型中是有用的，因为许多转基因和品系分离的小鼠品系可作为生物资源使用。因此，对于许多神经科学研究人员来说，为小鼠构建TMS-EEG组合测量系统的便捷方法是可取的。
本研究提出了一种TMS-EEG组合方法，可用于同时刺激和记录小鼠大脑，小鼠大脑是研究中使用的主要转基因动物类型，可以在典型的神经科学实验室中轻松构建。首先，描述了使用常规螺旋电极和柔性基板的低成本脑电图记录系统，以在每个实验中可重复地分配电极阵列位置。其次，磁刺激系统是使用毫米尺寸的线圈构建的，可以在典型的实验室中轻松定制。第三，TMS-EEG组合系统记录对声音和磁刺激的神经活动。本研究中提出的方法可以揭示在小动物中产生特定疾病的机制，并且可以翻译在动物模型中获得的结果以了解相应的人类疾病。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D printer</td>
			<td>Zhejiang Flashforge 3D Technology Co., Ltd</td>
			<td>FFD-101</td>
			<td>The printer used for 3D-printing the donut-shaped disks</td>
		</tr>
		<tr>
			<td>ATROPINE SULFATE&#160; 0.5 mg</td>
			<td>NIPRO ES PHARMA CO., LTD.</td>
			<td>-</td>
			<td>Atropine sulfate</td>
		</tr>
		<tr>
			<td>Bipolar amplifier</td>
			<td>NF Corp.</td>
			<td>KIT61380</td>
			<td>For amplifying waveforms for coil input</td>
		</tr>
		<tr>
			<td>Butorphanol</td>
			<td>Meiji Seika Pharma 
			Co., Ltd., Tokyo, Japan</td>
			<td>-</td>
			<td>For anathesis of animals</td>
		</tr>
		<tr>
			<td>Commercial manufacturer of flexible 2D array</td>
			<td>p-ban.com Corp.</td>
			<td>-</td>
			<td>URL: https://www.p-ban.com/</td>
		</tr>
		<tr>
			<td>Computer prograom to analyze output signals</td>
			<td>Natinal Instruments</td>
			<td>NI-DAQ and&#160; NI-DAQmx Python</td>
			<td>To analyze output signals from the hall-effect sensor</td>
		</tr>
		<tr>
			<td>Connector</td>
			<td>Harwin Inc.</td>
			<td>G125-FV12005L0P</td>
			<td>For connector to conect to the measuring system</td>
		</tr>
		<tr>
			<td>Copper pad</td>
			<td>p-ban.com Corp.</td>
			<td>copper</td>
			<td>Copper pad on each substrate</td>
		</tr>
		<tr>
			<td>Copper wire</td>
			<td>Kyowa Harmonet Ltd.</td>
			<td>P644432</td>
			<td>The windings of the coil</td>
		</tr>
		<tr>
			<td>DAQ board</td>
			<td>National Instruments Corp.</td>
			<td>USB-6343</td>
			<td>For measuring the magnitic flux density of the coil</td>
		</tr>
		<tr>
			<td>Dental cement</td>
			<td>SHOFU INC.</td>
			<td>Quick Resin</td>
			<td>Self-Curing Orthodontic Resin</td>
		</tr>
		<tr>
			<td>ECoG electrode</td>
			<td>NeuroNexus Inc.</td>
			<td>HC32</td>
			<td>For reference to design of the flexible 2D array</td>
		</tr>
		<tr>
			<td>Epoxy resin</td>
			<td>Konishi Co. Ltd.</td>
			<td>#16123</td>
			<td>For coil construction</td>
		</tr>
		<tr>
			<td>Ethyl Carbamate</td>
			<td>FUJIFILM Wako Pure Chemical Corp.</td>
			<td>050-05821</td>
			<td>For urethan anesthesia</td>
		</tr>
		<tr>
			<td>Flat ribbon cable</td>
			<td>Oki Electric Cable Co., Ltd.</td>
			<td>FLEX-B2(20)-7/0.1 20028 5m</td>
			<td>For cable to connect between surface-mount connector and measuring sysytem</td>
		</tr>
		<tr>
			<td>flexible substrate</td>
			<td>p-ban.com Corp.</td>
			<td>polyimide</td>
			<td>Baseplate of flexible substrate</td>
		</tr>
		<tr>
			<td>Function generator</td>
			<td>NF Corp.</td>
			<td>WF1947</td>
			<td>For generating waveforms for coil input</td>
		</tr>
		<tr>
			<td>Hall-effect sensor</td>
			<td>Honeywell International Inc.</td>
			<td>SS94A2D</td>
			<td>For measuring the magnitic flux density of the coil</td>
		</tr>
		<tr>
			<td>IDC crimping tool</td>
			<td>Pro&#39;sKit Industries Co.</td>
			<td>6PK-214</td>
			<td>To crimp the IDC and one end of the flat ribbon cable; Flat cable connector crimping tool</td>
		</tr>
		<tr>
			<td>Instant glue</td>
			<td>Konishi Co. Ltd.</td>
			<td>#04612</td>
			<td>For coil construction</td>
		</tr>
		<tr>
			<td>Insulation-displacement connector (IDC&#160;)</td>
			<td>Uxcell Japan</td>
			<td>B07GDDG3XG</td>
			<td>2 &#215; 10 pins and a 1.27 mm pitch&#160;</td>
		</tr>
		<tr>
			<td>LCR meter</td>
			<td>NF Corp.</td>
			<td>ZM2376</td>
			<td>For measuring the AC properties of the coil</td>
		</tr>
		<tr>
			<td>Manipulator</td>
			<td>NARISHIGE Group.</td>
			<td>SM-15L</td>
			<td>For manipulating the coil</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Kobayashi Kako, Fukui, Japan</td>
			<td>-</td>
			<td>For anathesis of animals</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Astellas Pharma, Tokyo, Japan</td>
			<td>-</td>
			<td>For anathesis of animals</td>
		</tr>
		<tr>
			<td>Miniature screw</td>
			<td>KOFUSEIBYO Co., Ltd.</td>
			<td>S0.6*1.5</td>
			<td>For EEG-senseing and reference electrode</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Japan SLC, Inc.</td>
			<td>C57BL/6J&#160;(C57BL/6JJmsSlc)</td>
			<td>Experimental animal</td>
		</tr>
		<tr>
			<td>Permalloy-45 rod</td>
			<td>The Nilaco Corp.</td>
			<td>780544</td>
			<td>The core of the coil</td>
		</tr>
		<tr>
			<td>Recording system</td>
			<td>Plexon Inc.</td>
			<td>OmniPlex</td>
			<td>For EEG data acquisition</td>
		</tr>
		<tr>
			<td>Stainless wire</td>
			<td>Wakisangyo Co., Ltd.</td>
			<td>HW-136</td>
			<td>For grasp by manipulator</td>
		</tr>
		<tr>
			<td>Stereotaxic apparatus</td>
			<td>NARISHIGE Group.</td>
			<td>SR-5M-HT</td>
			<td>To fix a mouse head</td>
		</tr>
		<tr>
			<td>Surface-mount connector</td>
			<td>Useconn Electronics Ltd.</td>
			<td>PH127-2x10MG</td>
			<td>For connector to mount on the flexible 2D array</td>
		</tr>
		<tr>
			<td>Testing equipment (LCR meter)</td>
			<td>NF Corp.</td>
			<td>ZM2372</td>
			<td>Contact check and impedance measurements</td>
		</tr>
		<tr>
			<td>White PLA filament</td>
			<td>Zhejiang Flashforge 3D Technology Co., Ltd</td>
			<td>PLA-F13</td>
			<td>The material used for 3D-printing the donut-shaped disks</td>
		</tr>
		<tr>
			<td>Xylocaine Jelly 2%</td>
			<td>Sandoz Pharma Co., Ltd.</td>
			<td>-</td>
			<td>lidocaine hydrochloride</td>
		</tr>
	</tbody>
</table>

low-cost electroencephalographic recording system combined with a millimeter-sized coil to transcranially stimulate the mouse brain in vivo

这里提出了一种低成本的脑电图（EEG）记录系统，利用毫米大小的线圈 驱动 体内小鼠大脑的经颅磁刺激（TMS）。使用传统的螺旋电极与定制的、灵活的多电极阵列基板相结合，可以从小鼠大脑进行多位点记录。此外，我们还解释了如何使用实验室中常见的低成本设备生产毫米尺寸的线圈。还介绍了制造柔性多电极阵列基板的实用程序和螺杆电极的手术植入技术，这是产生低噪声脑电信号所必需的。尽管该方法对于从任何小动物的大脑中记录是有用的，但本报告侧重于麻醉小鼠头骨中的电极实施。此外，这种方法可以很容易地扩展到清醒的小动物，它通过公共适配器 与 系留电缆连接，并在录制过程中用TMS设备固定在头部。描述了当前版本的EEG-TMS系统，其最多可以包括32个EEG通道（具有16个通道的设备作为具有较少通道的示例）和一个TMS通道设备。此外，简要报告了通过将EEG-TMS系统应用于麻醉小鼠获得的典型结果。

Transcranial magnetic stimulation (TMS) is a promising tool for human brain science, clinical application, and animal model research because of its non-/low invasiveness. During the early stage of TMS applications, measurement of the cortical effect in response to single- and paired-pulse TMS in humans and animals was restricted to the motor cortex; easily measurable output was limited to motor evoked potentials and induced myoelectric potentials involving the motor cortex1,2. To expand the brain regions that can be measured by TMS modulation, electroencephalographic (EEG) recording was integrated with single- and paired-pulse TMS as a useful method to directly examine the excitability, connectivity, and spatiotemporal dynamics of areas throughout the whole brain3,4,5. Thus, the simultaneous application of TMS and EEG recording (TMS-EEG) to the brain has been used to probe various superficial cortical brain areas of humans and animals to investigate intracortical neural circuits (see Tremblay et al.6). Moreover, TMS-EEG systems can be used to examine additional cortical spatiotemporal characteristics, including the propagation of signals to other cortical areas and the generation of oscillatory activity7,8.
However, the mechanism of action of TMS in the brain remains speculative because of the non-invasiveness of TMS, which limits our knowledge of how the brain functions during TMS applications. Therefore, invasive translational studies in animals ranging from rodents to humans are of crucial importance to understand the mechanism of the effects of TMS on neural circuits and their activity. In particular, for combined TMS-EEG experiments in animals, a simultaneous stimulation and measurement system has not been intensively developed for small animals. Therefore, experimentalists are required to construct such a system by trial and error according to their specific experimental requirements. In addition, mouse models are useful among other in vivo animal species models because many transgenic and strain-isolated mice strains are available as biological resources. Thus, a convenient method to build a TMS-EEG-combined measurement system for mice would be desirable for many neuroscience researchers.
This study proposes a TMS-EEG-combined method that can be applied for simultaneous stimulation and recording of the mouse brain, which is the main type of transgenic animal used in research, and that can easily be constructed in typical neuroscience laboratories. First, a low-cost EEG recording system is described using conventional screw electrodes and a flexible substrate to reproducibly assign an electrode-array position in each experiment. Second, a magnetic stimulation system is constructed using a millimeter-sized coil, which can easily be custom-made in typical laboratories. Third, the TMS-EEG-combined system records neural activity in response to sound and magnetic stimulation. The method presented in this study can reveal the mechanisms that generate specific disorders in small animals, and the results obtained in the animal models can be translated to understand the corresponding human disorders.

作者聚焦： 低成本脑电图记录系统结合毫米级线圈经颅刺激小鼠体内大脑  

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

In neuroscience, TMS and EEG recording are frequently used to advance our understanding of the brain. TMS is especially promising due to its non-invasiveness. Combining TMS with EEG recording enables researchers to explore excitability, connectivity, and spatiotemporal dynamics across various brain regions.This protocol aims to develop a simultaneous stimulation measurement system for TMS-EEG experiments with small animals. These system have not been extensively developed, forcing researchers to create a system to trial and error according to the unique experimental requirements. This easily adaptable approach can be set up in standard neuroscience labs, making it more accessible and practical for researchers.The findings of this study will advance research and elucidating mechanisms behind specific disorders in small animals, which are essential models for human candidates. Further, these models will improve their understanding of human conditions and help guide the creation of more effective treatments and interventions.

在麻醉的C57BL / 6J小鼠中记录的样品脑电图数据，柔性基质与螺旋电极相结合，如下所示。
作为一个典型的例子，在具有相同刺激的60项试验中，显示了响应声音刺激（8 kHz音爆，80 dB声压级[SPL]）而产生的平均EEG波形（图4A）。记录通道映射的原理图也显示在图4A的中间。来自CHS 5,7,10和12的反应是从两个颞叶的听觉皮层附近区域记录?...

Watch this Scientific Journal Video about Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo at JoVE.com

提出了一种低成本的脑电图记录系统，结合毫米尺寸的线圈来驱动体内 小鼠大脑的经颅磁刺激。使用具有定制、柔性、多电极阵列基板的传统螺旋电极，可以从小鼠大脑响应经颅磁刺激进行多位点记录。

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

在神经科学中，TMS和EEG记录经常被用来促进我们对大脑的理解。TMS由于其非侵入性而特别有前途。将TMS与EEG记录相结合，使研究人员能够探索不同大脑区域的兴奋性，连通性和时空动力学。该协议旨在开发用于小动物TMS-EEG实验的同步刺激测量系统。这些系统尚未得到广泛开发，迫使研究人员根据独特的实验要求创建一个试错系统。这种易于适应的方法可以在标准的神经科学实验室中建立，使其对研究人员来说更容易获得和实用。这项研究的结果将推进研究和阐明小动物特定疾病背后的机制，这是人类候选者的基本模型。此外，这些模型将提高他们对人类状况的理解，并有助于指导创造更有效的治疗和干预措施。

低成本脑电图记录系统结合毫米级线圈，在体内经颅刺激小鼠大脑

Abstract