This work was supported by the Murata Science Foundation, the Suzuken Memorial Foundation, the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, and&#160;a Grant-in-Aid for Exploratory Research (grant number 21K19755, Japan) and for Scientific Research (B) (grant number 23H03416, Japan) to T.T.

In the present study, all animal experiments were performed following the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval from the Institutional Animal Care and Use Committee of Hokkaido University. C57BL/6J mice, two male and three female, 8 to 10 weeks old, were used for the present study. This is a terminal procedure. The animals were obtained from a commercial source (see Table of Materials).
1. Flexible two-dimensiona...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

<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>

The authors have nothing to disclose.

This study addresses a multi-site EEG recording system combined with a magnetic stimulation system designed for small animals, including mice. The constructed system is low-cost and easily constructed in physiological laboratories, and can extend their existing measurement setups. The surgical procedure necessary to obtain data from the mouse recording system is profoundly simple if such laboratories have previous experience with standard electrophysiological experiments.
One advantage of usin...

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.

<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

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

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.

Sample EEG data recorded in anesthetized C57BL/6J mice with the flexible substrate combined with the screw electrodes are presented below.
As a typical example, the average EEG waveforms generated in response to sound stimulation (8 kHz tone-burst, 80 dB sound pressure level [SPL]) are shown for 60 trials with identical stimuli (Figure 4A). A schematic of recording channel mapping is also presented in the middle of Figure 4A. The resp...

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

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

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

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Neuroscience

Electroencephalographic (EEG) Electrode Implantation on Mouse Brain

To begin, use an intraperitoneal anesthetized mouse and expose the skull Using standard surgical procedures. Attach the stainless steel rod mounted to the 2D electrode array on the backside of the flexible substrate to a manipulator. Then place the flexible substrate on the exposed skull.Adjust the location of channels 3 and 14 on the array to fit within the inferior colliculus. Draw four small circles at the locations of channels 3, 8, 9, and 14 on the skull with a permanent marker to serve as targeting landmarks. Next, dry the skull surface to improve the adherence of the dental cement and to electrically isolate the 2D electrode array on the flexible substrate from the mouse skull.Apply a layer of dental cement to the skull surface approximately one millimeter thick and allow the cement to cure for approximately 30 minutes. Align the flexible substrate in line with the small circular marks on the surface of the skull. Then position the tip of a dental drill on each electrode pad hole on the flexible substrate and carefully drill into the skull.Using a screwdriver, secure the miniature screw electrodes into the drill holes of the skull. Crimp the head of the screw electrode and the electrode pad firmly. Finally, measure the conductance between each screw electrode and the connector with testing equipment, such as an LCR meter to confirm electrical conductivity.

Electroencephalographic (EEG) Signal Recording System and Procedure

1.7K Views.  Hokkaido University.  A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

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