This work was supported in part by the GIST Research Institute (GRI), the GIST-Caltech Research Collaboration Project through a grant provided by GIST in 2017. Also supported by research grant (NRF-2016R1A2B4015381) of the National Research Foundation (NRF) funded by the Korean government (MEST), and by KBRI basic research program through Korea Brain Research Institute funded by the Ministry of Science, ICT, and Future Planning (17-BR-04).

Animal care and handling followed the institutional guideline of the Gwangju Institute of Science and Technology (GIST).
NOTE: The procedure for acquiring the VEP signal from a mouse in vivo consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing.
1. Sensor Assembly
<ol>
	<li>Prepare sixteen pins for one non-invasive electrode. 
	NOTE: Each pin-type electrode consists of three parts: a probe...

<ol>
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The authors have nothing to disclose.

We firstly focused on the design of the sensor, prioritizing practicality by minimizing complex surgical procedures. The deformable EEG sensor is comprised of sixteen pins: fourteen for recording, one for ground, and the last one for reference electrodes. Each electrode has the plunger-spring-barrel structure, which applies deformability onto the electrode's contact surface, so they facilitate uniform and stable signal acquisition from the curved and tender mouse's scalp. Considering the welfare of the animals, we tried ...

As the number of patients with senile degenerative brain diseases such as dementia, Alzheimer&#39;s, Parkinsonian syndromes, and stroke have increased with an aging population and an increasing life expectancy, the long-term societal burden of these diseases has also increased1,2,3. In addition, most neurodevelopmental diseases, such as Schizophrenia and autism, are accompanied by cognitive and behavioral disorders which affect a patient&#39;s whole life2,3,4. For this reason, researchers have been struggling to improve the diagnosis, prevention, pathological understanding, long-term observation, and treatment of brain diseases. However, problems remain stemming from the brain&#39;s complexity and unrevealed disease pathologies. Translational research may be a promising tool for identifying solutions because it enables the transfer of basic research to clinical applications within a shorter time frame, at lower cost, and with a higher success rate in neuroscience fields5,6,7. Another goal of translational research is to examine applicability in human subjects, which requires non-invasive experimental approaches in animals that allow comparisons to the same method for humans. These conditions have led to several significant needs for developing non-invasive animal preparation methods. One method is electroencephalography (EEG), which reveals cortical brain connectivity and activity two-dimensionally with high temporal resolution, and which benefits from a non-invasive protocol. The event-related potential recording (ERP) is one of the typical experimental paradigms that utilize EEG.
Numerous previous studies employed non-invasive EEG methods for targeting humans subjects, whereas invasive methods, such as implant screws and pole type electrodes, have been used in animal studies8,9,10,11,12. The signal quality and characteristics of these methods are significantly reliant on the invasiveness of the sensor placement. For successful translational research, Garner emphasized using the same conditions for animal study as those used for human research13. For basic research using animals, however, non-invasive EEG methodologies are not prevalent. A novel approach using a non-invasive scalp EEG sensor system focusing on laboratory mice would be a reliable and efficient tool for translational research that can be applied to the non-invasive paradigms for human beings, as well.
Numerous mouse EEG studies led the way by commercializing PCB (printed circuit board) based multi-channel electrodes14,15,16. Although they adopted an invasive method, they had a restricted number of channels (3-8), which made it harder to observe large-scale brain dynamics. Furthermore, applications can be restricted by their invasiveness and high cost. In another research study, the KIST (Korea Institute of Science and Technology) developed a 40 channel polyimide-based thin-film electrode and attached it to a mouse&#39;s skull17,18,19,20. This work acquired the highest number of mouse EEG channels. It was, however, mechanically weak and not easy to reuse; therefore, it was inappropriate for long-term observations, leading to a weakened signal, possibly caused by an immune reaction. Meanwhile, Troncoso and M&#233;gevand acquired a sensory evoked potential (SEP) on rodents&#39; skulls with thirty-two stainless steel electrodes secured by a perforated Poly(methyl methacrylate) (PMMA, acrylic glass) grid21,22,23. Despite their high signal quality, the electrodes were mechanically flexible and tender; therefore, they had difficulties being applied to multiple experiments. In addition, this method was still minimally invasive. Although these methods provide good signal quality, the surface area of a mouse&#39;s skull is limited, therefore the number of electrodes is restricted using a stainless pole-type electrode. A number of previous EEG studies for mice showed several limitations. In this study, we will show a new method for measuring EEG applicable in pre-clinical translational research using a non-invasive dry-type multi-channel sensor.
In order to overcome the limitations of previous animal EEG methodologies, which included the intrinsic complexity of animal preparation, invasiveness, high cost, wastefulness, and weak mechanical strength, we sought to develop a new electrode which exhibits flexibility, dry type status, multi-channel capabilities, non-invasiveness, and re-usability. In the following protocol, we will describe the process of measuring visual evoked potential (VEP) recordings on a mouse scalp using a dry, non-invasive, multi-channel EEG sensor. This method utilizes easily available resources, therefore lowering the barrier to entry into animal experimentation in the biomedical engineering field.

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			<td>Xylazine hydrochloride 23.32mg/mL</td>
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			<td>NaCl 9 g/1000 mL</td>
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			<td>amplifier</td>
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			<td height="42" style="height:42px;width:260px;">&#160;441 style straight body Touch Proof connector</td>
			<td style="width:173px;">PlasticsOne</td>
			<td style="width:143px;">441000PSW080001</td>
			<td>441 - 000 PSW 80&#34; (BLACK)</td>
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visual evoked potential recordings in mice using a dry non-invasive multi-channel scalp eeg sensor

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable because of the plunger-spring-barrel structural facet and mechanical strengths resulting from metal materials. The whole process for acquiring the VEP responses in vivo from a mouse consists of four steps: (1) sensor assembly, (2) animal preparation, (3) VEP measurement, and (4) signal processing. This paper presents representative measurements of VEP responses from multiple mice with a submicro-voltage signal resolution and sub-hundred millisecond temporal resolution. Although the proposed method is safer and more convenient compared to other previously reported animal EEG acquiring methods, there are remaining issues including how to enhance the signal-to-noise ratio and how to apply this technique with freely moving animals. The proposed method utilizes easily available resources and shows a repetitive VEP response with a satisfactory signal quality. Therefore, this method could be utilized for longitudinal experimental studies and reliable translational research exploiting non-invasive paradigms.

We calculated the ensemble average of VEP responses from eleven mice as shown in Figure 2. This result shows the VEP responses obtained through this experiment from the pre-stimulation period (-300 ms) to the post-stimulation period (600 ms), as the stimulation is given at time 0 s. It is noticeable that the signal fluctuates only for a while (less than 300 ms) after the stimulation, while the signal steadily stabilizes over time during the post-stimulation p...

Watch this Scientific Journal Video about Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor at JoVE.com

Visual Evoked Potential Recordings in Mice Using a Dry Non-invasive Multi-channel Scalp EEG Sensor

We designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable. This paper describes the whole process from manufacturing the proposed EEG electrode to signal processing of visual evoked potential (VEP) signals measured on a mouse scalp using a dry non-invasive multi-channel EEG sensor.

For scalp EEG research environments with laboratory mice, we designed a dry-type 16 channel EEG sensor which is non-invasive, deformable, and re-usable ...

The overall goal of this procedure is to measure visual evoked potentials on a mouse scalp using a dry, non-invasive, and multi-channel EEG sensor. This method can help answer quick questions in pre-clinical translational research field to breach the signal gap in non-invasive EEG research between human and laboratory animals. The main advantage of this technique is it's non-invasiveness, which enhances comprehension of animal EEG.Furthermore, it provides safe and convenient experimental environments. Begin by preparing 16 pins for one non-invasive electrode. Cut two pieces of glass fiber substrates with the size of 15 millimeters by 17 millimeters.Next, make 16 1.2 millimeter diameter holes using a precision engraving machine. Then, drill 16 holes into the flat substrates according to the probe coordination, evenly at an interval of two millimeters. Stack two substrates, and apply one drop of fast-acting adhesive glue between the substrate layers to create a double layer of three millimeter thickness, supporting 16 stable and parallel electrodes during signal acquisition.Then, manually assemble the 16 electrodes onto the substrate one by one. Solder and link each electrode's ending solder cap part to the touch-proof connector. Finally, cover and hide the exposed junctions with heat-shrink tubing for electrical insulation.On the anesthetized mouse, begin by applying eye ointment with a cotton swab to keep the cornea moist. Remove the hairs around the head and shoulders with a hair clipper. Then, spread commercially available depilatory cream, and keep it on this area for three to four minutes.Finally, remove the applied depilatory with a spatula, and then wipe up the rest with wet wipes, applying water several times. Begin by mounting the mouse's head onto the stereotaxic frame by placing ear bars into the mouse's ear canals, and tightening them in place. Mount the sensor into the custom-made electrode holder.Then, fix the sensor holder onto the stereotaxic frame. Locate the flexible electroencephalography, or EEG sensor, taking into account the reference electrode and bregma position. Then, carefully lower the sensor in the vertical direction, so that the arrayed electrode plungers contact the mouse's scalp evenly onto the curved margin.Check that the impedances are within the proper range, from 100 kiloohms, to two megaohms. Reposition the electrode when any impedance value is out of the range. Next, position the photostimulator 20 centimeters away from the mouse's eyes.Set the parameters of the experimental devices such that the sampling frequency is 500 hertz, notch filtering is 60 hertz, the inter-stimulus interval at 10 seconds, flash duration at 10 milliseconds, and the number of flash stimuli at 100 trials per subject. Finally, before starting the EEG recordings, adapt the mouse for 10 minutes in the dark cage for dark visual adaptation. Average visual evoked potential responses indicate minimal signal fluctuation after stimulation, less than 300 milliseconds in duration.The signal steadily stabilizes over time during the post-stimulation period. Further, 14 channels can be categorized into several groups based on the VEP responses, revealing similar morphologies and patterns. Once mastered, this technique can be completed in one hour if it's performed properly.While attempting this procedure, it's important to remember to check the animal's status of anesthesia. Combining this procedure with other methods, like brain stimulation or surface-deep electrophysiological recordings can be performed in order to answer additional questions, like brain stimulation parameter optimization, or source localization. After its development, this technique paved the way for researchers in the field of translational research areas to connect basic science research to human brain studies with comparable, reliable, and efficient research without any invasive surgical preparation.After watching this video, you should have a good understanding of how to measure visual evoked potentials on a mouse scalp using a new electrode that has the benefit of flexibility, dry type status, multichannel capabilities, non-invasiveness, and re-usability.

visual-evoked-potential-recordings-mice-using-dry-non-invasive-multi

Research

JoVE Journal

Neuroscience

8.8K Views.  Gwangju Institute of Science and Technology (GIST). The overall goal of this procedure is to measure visual evoked potentials on a mouse scalp using a dry, non-invasive, and multi-channel EEG sensor. This method can help answer quick questions in pre-clinical translational research field to breach the signal gap in non-invasive EEG research between human and laboratory animals. The main advantage of this technique is it's non-invasiveness, which enhances comprehension of animal EEG.Furthermore, it provides safe and convenient experiment...