This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under the Emmy-Noether program, BL 1591/1-1 - Project ID 411333557. We thank Suong Nguyen, Manuela J&#228;ger, and Maria Stollmann for their assistance in filming the video. We thank Joanna Scanlon for the video voiceover.

The general procedure used in this protocol was approved by the ethics board of the University of Oldenburg. The participant provided written informed consent prior to their participation.
NOTE: The c-grids should only be used on undamaged skin and with participants that do not have an allergy to the adhesive used. It has two sides.&#160;There is black text on the outside. The conductive surfaces of the electrodes are on the inside, and they face the skin of the participant during the recording. Importantly, handle these grids with care. Do not touch the conductive surfaces, do not fold the grids, do not excessively bend them, and avoid pulling on them.
1. Testing
NOTE: If handled with care, c-grids can be reused several times. To ensure optimal functioning, check that all the electrodes are working properly before the next recording. Perform the same procedure for new grids to identify potential problems (e.g., due to problems in the manufacturing process) before the recording starts. There are several options to quickly check for problems (e.g., a broken electrode).
<ol>
	<li>Option 1: Multimeter.
	<ol>
		<li>Set a multimeter to measure resistance.</li>
		<li>Attach one pin of the multimeter to the electrode and the other pin to the corresponding contact on the connector end.</li>
		<li>Check if a low resistance (&#60;10 k&#8486;) can be measured for each electrode.</li>
	</ol>
	</li>
	<li>Option 2: Electrode gel
	<ol>
		<li>Use electrode gel to bridge all the electrodes. Make sure that there are no gaps between the electrodes.</li>
		<li>Attach the grid to the connector of an amplifier. To see a signal, attach the grid to the side with the reference and ground electrodes according to the connector layout that is used.</li>
		<li>Use the impedance check of the amplifier. Check the impedance of the reference electrode and all eight recording electrodes (10 electrodes in total minus the ground and reference electrodes); they must all have a low impedance (&#60;10 k&#8486;). Afterward, wipe off the gel.</li>
	</ol>
	</li>
	<li>Option 3: Water 
	NOTE: Use this option with caution in order to not cause any water damage to the equipment.
	<ol>
		<li>Submerge all the electrodes in a glass of water, but make sure to keep the tail of the grid dry. Alternatively, place the c-grid in a plate filled with water (with the electrodes facing the plate).</li>
		<li>Attach the grid to the connector of the amplifier.</li>
		<li>Use the impedance check of the amplifier. Check the impedance of the reference electrode and all eight recording electrodes (10 electrodes in total minus the ground and reference electrodes); they must all have a low impedance (&#60;10 k&#8486;). Afterward, dry the c-grid with a tissue.</li>
	</ol>
	</li>
</ol>
2. Preparing the participant
NOTE: For high-quality recordings, the participant should have clean and dry hair, should not have used any hair products (e.g., styling products) or skin products, and should not be wearing make-up. If possible, the participants should wash their hair directly before the recording with a mild and neutral shampoo and also wash the areas around the ears. Ask the participants to indicate if any of the preparatory steps are uncomfortable for them.
<ol>
	<li>To prepare the participant, the experimenter needs access to the area behind and around the ear. For participants with longer hair, use hair clips for easier access.</li>
	<li>Place a c-grid around the participant&#39;s ear to see how it fits. Additionally, check if it can be positioned around the ear without touching the ear. Ensure it does not touch the back of the ear or the ear lobe, as this can be uncomfortable after some time. This pre-fitting also gives an indication of the area that will be covered and, thus, needs to be cleaned. 
	NOTE: These grids come in one size and do not fit all ear sizes. For larger ears, cut some of the plastic around the electrodes on the inside of the C with a small pair of scissors. Pay special attention to not cut into the electrodes or the conductive path.</li>
	<li>Apply a small drop of abrasive electrode gel onto a tissue. Use the gel to clean the skin around the participant&#39;s ear with some pressure, but make sure it remains comfortable for the participant. Make sure to generously clean the whole area that will be covered.</li>
	<li>Dip a tissue in some alcohol, and clean the area behind the ear with this tissue.</li>
	<li>Dry the cleaned area with a clean towel.</li>
	<li>For higher levels of comfort, optionally place a small piece of tape on the back side of the ear.</li>
	<li>Repeat all of the above steps (steps 2.1-2.5) for the other ear.</li>
</ol>
3. Preparing and fitting the grids
NOTE: There are different ways of attaching the c-grid using double-sided tape. Presented here are two options: c-shaped stickers (provided by the manufacturer) that cover the whole surface and small circular stickers that are placed individually around the electrodes (e.g., when reusing).
<ol>
	<li>Attach double-sided adhesive stickers (either the c-shaped or individual stickers) around each electrode. Make sure that the stickers do not cover the conductive surface of the electrodes.</li>
	<li>Put small drops (lentil-sized) of electrode gel on each electrode. Avoid using too much gel, as this might spill onto the adhesive material and reduce the adhesion to the skin. Too much gel could also create bridges between the electrodes.</li>
	<li>Remove the cover of the adhesive sticker(s). Reapply the gel in case it was removed during this step. Alternatively, remove the first cover, and apply the gel then; however, this requires a very steady hand so the gel does not spill accidently onto the adhesive.</li>
	<li>Ask the participant to hold their hair away from the ear so that it does not obstruct the fitting. Move any hair out of the way as much as possible so that the stickers touch the skin directly. Depending on the hairline, this is not always possible (e.g., when there is hair directly above the ear).</li>
	<li>Position the grid around the ear, and when in place, press it into the skin. Make sure to not place it too close to the ear, as this may become uncomfortable for the participant. Leave some space (1 mm to 2 mm) between the grid and the back side of the ear. Additionally, ask the participant to press onto the electrodes.</li>
	<li>Repeat all of the above steps (steps 3.1-3.5) for the other ear.</li>
	<li>Remove any hair clips. Carefully place glasses or the strips of facial masks on the ears if necessary.</li>
</ol>
4. Connecting
<ol>
	<li>Connect the connector to the amplifier. During this step, avoid excessively bending or pulling on the c-grid.</li>
	<li>Plug the contacts into the connector. Make sure that the contacts are plugged in on the correct side. Ensure that the exposed contacts on the inside of the c-grid face the contacts in the connector. 
	NOTE: It is important to know the layout of the connector being used (including the position of the ground and the reference electrodes). Depending on the system being used, the layout may differ. To build a connector, visit&#160;https://uol.de/psychologie/abteilungen/ceegrid. With the right connector, c-grids can be connected to any amplifier.</li>
	<li>To hold the amplifier in place, use a headband, for example, to fixate it on the head. 
	&#8203;NOTE: The Oldenburg lab uses an amplifier that is built into a neck speaker called nEEGlace. The nEEGlace makes the setup more comfortable and faster.</li>
</ol>
5. Check the impedance and data
<ol>
	<li>Connect the amplifier to a smartphone (optionally: a laptop) via Bluetooth.</li>
	<li>Check the impedance of the electrodes with the impedance check of the amplifier. The impedance usually improves over time (5 min to 10 min) and does not have to be below 10 k&#8486; for each electrode in the beginning. Do not try to put more gel underneath electrodes with a high impedance.</li>
	<li>Check the EEG signal. Ask the participant to clench their jaws, to blink, and to close their eyes (alpha activity). Observe the corresponding artifacts and alpha activity in the signal. Make sure every electrode provides a good signal. If the resulting EEG signal is poor, remove the grid, wipe off any residual gel around the ear of the participant, and fit a new one.</li>
	<li>Begin recording.</li>
</ol>
6. Removing and cleaning up
<ol>
	<li>After finishing the data recording, disconnect the phone (or laptop) from the amplifier. Detach the grids from the amplifier, and remove the amplifier from the participant. Gently remove the c-grids from the participant. Make sure to neither bend the c-grid too much nor to pull out the hair of the participant. Allow the participants to clean themselves with tissues or a towel.</li>
	<li>Soak the grids in water for some minutes. They can be submerged completely.</li>
	<li>Carefully detach the stickers to avoid damage. Rinse off any remaining gel. Air-dry the grids. Do not rub over the conductive surface of the electrodes.</li>
	<li>Store the c-grids safely in a dark and dry place.</li>
</ol>

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The authors report no conflicts of interest.

Provided here is a protocol for ear-EEG recordings with the c-grids. Following the steps of this protocol ensures high-quality recordings. In the following paragraphs, a comparison is made with cap-EEG, the most critical steps in the protocol along with some best practice recommendations are discussed, and some modifications are discussed.
Comparison of c-grids to cap-EEG and in-ear EEG 
The c-grid allows the unobtrusive recording of brain activity in everyday life settings and is well suited for longer recordings. It has several advantages compared to cap-EEG. First, due to its weight, comfort, and low visibility, it barely restricts participants in their everyday activities1. Second, it can be worn for extended periods of time - more than 11 hours in one study6- without the electrodes falling off1,3,6, since they are sealed by the adhesive stickers. On the downside, the c-grid covers only a fraction of the surface of cap-EEG and, thus, cannot replace cap-EEG for all purposes. However, in cases when a lightweight, unobtrusive, quick-to-setup, minimally restricting solution is necessary (e.g., in the workplace), c-grids can provide relevant neural information.
The comparison of results across participants is potentially more difficult for c-grids compared to cap-EEG. For cap-EEG, often the international 10-20 system is used to facilitate the comparison of results across studies and across participants with different head sizes. In this system, the electrodes are positioned relative to specific anatomical landmarks (i.e., the nasion and inion for front to back and the ears for left to right). In practice, different cap sizes are used to account for different head sizes and thereby approximate the optimal electrode positioning. The c-grid cannot be easily integrated into that system for two reasons. First, these are currently available in one size and, thus, cover more or less space depending on the head size. Second, the shape of the ear influences the positioning of the grids. In general, the two top-most electrodes will be directly above the ear, but depending on the ear shape, they might be tilted more to the front or to the back. We are not aware of any study that has investigated whether these shifts in electrode positions are large enough to be of relevance.
Another approach to measuring&#160;ear-EEG is to place the electrodes inside the ear, for instance, in the outer ear canal or the concha23,24,25. Such an approach offers even lower visibility than the c-grid but leads to recording signals with lower amplitudes due to the small distances between the electrodes26.
Most critical steps 
EEG in general, and especially mobile ear-centered EEG, remains a challenging technology. Therefore, the careful preparation of the participant and placement of the grids is essential to ensure good data quality over time. The preparation starts with the hair and the skin of the participants. The hair and skin around the ear should be washed and dried. In addition to that, the experimenter needs to carefully clean the area around the ear with abrasive gel and alcohol and ensure that the grids are firmly attached with the adhesive stickers. These steps are important and should be performed carefully to ensure good electrode-skin adhesion and a low impedance for longer periods. The skin cleaning especially can make the difference between a successful and an unsuccessful recording.
Even with proper care, however, the impedance for individual electrodes may still be poor directly after the placement of the electrodes. In general, the electrode-skin interface stabilizes over time, and we often observe that the impedance decreases within 5 min to 15 min. If the signal quality remains poor, it is recommended to completely remove the grids, wipe off any residual gel around the participant&#39;s ear, and fit a new one. It is faster to fit a new one as opposed to cleaning and preparing the previously removed grid. It is not recommended to add electrode gel to individual electrodes once the grid is fitted as this can compromise the adhesion strength of the stickers and can even lead to the bridging of the neighboring electrodes.
After the grid has been placed and when the impedance of the electrodes is low, data recording can begin. For longer recordings (&#62;1 h), a brief data quality check should be conducted at the beginning. For instance, a 3 min auditory oddball task is exemplified in this study, which can be conducted and analyzed quickly to ensure a good signal quality.
In some cases, recording with the c-grid might not be possible at all, such as when the grid is too small for the ear (even after cutting) or when the hairline is too close to the ear, meaning that the grid does not stick to the skin. If the grid &#34;hovers&#34; over some hair, researchers cannot expect high-quality data.
Troubleshooting 
Bad impedance and/or signal 
To avoid these problems, it is imperative that the skin is carefully cleaned before fitting. Additionally, one should make sure to test the functionality of each electrode before the fitting. For example, one should check that the grid is plugged in correctly into the connector and that each electrode has firm contact with the skin and then wait for a few minutes until the impedance and the signal improve. To further check the functionality after fitting, the individual electrodes should be pressed, and the resulting signal should be checked. If the corresponding signal of each electrode shows a response, the electrode is functional in principle. If all of the above steps do not help, one should remove the grid, wipe off the residual gel around the participant&#39;s ear, and fit a new one.
Situations with no signal 
Firstly, one should make sure the grid is connected properly to the amplifier, as well as ensuring that the grid connector is not upside down. There will be a signal only if the ground and reference electrodes are connected; whether the ground and reference will be on the left, the right, or on both sides depends on the connector.
Signal getting worse during the recording 
There may be several reasons for this issue that need to be addressed. First, some of the electrodes might have become detached from the skin. This can happen when the adhesive is compromised by the remains of the electrode gel, by hair under the electrodes, or due to interference from the participant (e.g., scratching around the ear or adjusting glasses). Second, there might be problems with the connection between the grid and the amplifier (i.e., the grid might have been pulled out of the amplifier, or its position might have shifted). Finally, the grid might have sustained damage during usage. This can happen if the tail of the c-grid is bent too strongly.
Channels showing identical signals 
In this case, the electrodes are bridged. One should remove the grid, wipe off the residual gel around the participant&#39;s ear, and fit a new one. One should also make sure to only use lentil-sized drops of electrode gel on each electrode to avoid bridging.
Participants reporting that the placement is uncomfortable 
The most common reason for decreased comfort is that the grid is placed too close to the backside of the ear. One should make sure to leave 1 mm to 2 mm between the c-grid and the back side of the ear. A small piece of tape attached behind the ear helps to increase comfort.
Modifications of the method 
The c-grid comes in one size. However, it allows for some flexibility regarding its size. By cutting the plastic of the inner side, the size may be reduced to fit larger ears. One should pay special attention in order to not cut into the electrodes or the conductive paths.
Depending on the amplifier used and the recording scenario, there are different ways to place the amplifier on the body. The fixed length of the tail of the grid and the fact that it points horizontally away from the ear limits the possible locations for placing the connector of the amplifier. Different manufacturers provide adapter cables that connect the grid to a specific amplifier (either mobile or lab-based). Different solutions have been proposed for placing the amplifier; some researchers use a headband3, whereas others integrate it into a basecap27. For shorter experiments, a headband is suitable. For longer experiments, the amplifier can be taped to the clothes6 or body2, stored in custom-made straps, taped to headphones worn around the neck1, or taped to a neck protector commonly used for mountain biking. We have developed a prototype that combines a neck speaker (for presenting auditory stimuli) with a mobile EEG amplifier and connectors to the c-grid (building instructions can be found here:&#160;https://github.com/mgbleichner/nEEGlace). We have used this approach successfully in a recent study (in preparation) in which we recorded ear-EEG for 4 h while participants worked in an office.
Future applications 
The c-grid is a promising tool for long-term recordings in everyday life. For instance, one can use it to investigate sound processing in everyday life1. With long-term recordings, circadian variations in cognition and auditory function can also be investigated28,29. For diagnostic purposes, the grid can be used for the long-term monitoring of epileptic seizures2, sleep staging6, or for measuring attention for hearing devices7,11.
Conclusion 
This protocol comprehensively equips researchers for experimenting with these c-grids in and beyond the lab. If researchers follow this protocol and carefully perform the steps, including the most important ones, such as skin cleaning and fitting the c-grid, they can expect high-quality data for their ear-EEG experiments.

With mobile ear-electroencephalography (EEG), brain activity can be recorded in everyday life, and new insights into neural processing beyond the lab can be gained1. To be suitable for everyday life, a mobile ear-EEG system should be transparent, unobtrusive, easy to use, motion-tolerant, and comfortable to wear even for several hours2. The c-grid (sold under the name cEEGrid), a c-shaped ear-EEG system, aims to meet these requirements to minimize interference with natural behavior. The grid consists of 10 Ag/AgCl electrodes printed on Flexprint material3. Combined with a miniaturized, mobile amplifier and a smartphone for data acquisition4,5, these grids can be used to collect ear-EEG data for more than 8 hours1,6.
Several studies conducted in the lab have shown the potential of c-grids for studying auditory and other cognitive processes. C-grids have been successfully used for auditory attention decoding with accuracies above chance level7,8,9,10,11. Segaert et al.12 used these arrays to quantify language impairment in patients with mild cognitive impairment. Garrett et al.13 showed that these arrays can capture auditory brain potentials originating from the brain stem. Apart from the research focusing on the auditory domain, Knierim et al.14 used the grids to investigate flow experiences (i.e., the sensation of total involvement in a task), as measured by changes in alpha power. Finally, Pacharra et al.15 used these grids for a visual task. All of these lab-based studies showcase the various cognitive processes that can be captured with these grids.
These grids can also be used for EEG recordings beyond the lab, as illustrated by several studies. For example, these arrays have been used to evaluate mental load in a driving simulator16,17 and to study inattentional deafness, the non-perception of critical alarm sounds, in a flight simulator18. The grids are especially promising for long-term recordings, such as the long-term monitoring of epileptic seizures2 and sleep staging6. H&#246;lle et al.1 used these grids to measure auditory attention during an office day for 6 hours. In sum, all of these studies highlight their potential to investigate various brain processes in and outside of the lab.
Every EEG recording requires careful preparation to obtain valid results. This is especially important for mobile applications where more artifacts can be expected than in the lab due to the movement of the participant. To ensure optimal results, specific preparation steps are necessary. We state the critical steps in preparing the grids, preparing the participant for data collection, and fitting and connecting the grids for EEG recordings. We point out potential mistakes and show examples of poor data quality when the attachment is not proper. Finally, representative results of a piano-played&#160;oddball task are shown.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Abrasive gel: Abralyt HiCl</td>
			<td>easycap GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AFEx app</td>
			<td>University of Oldenburg, Germany</td>
			<td></td>
			<td>for our exemplary data recording, open-source: https://zenodo.org/record/5814670#.Y0AavXZByUk</td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Carl Roth GmbH + Co. KG, Germany</td>
			<td></td>
			<td>70% isopropanol, 30% destilled water</td>
		</tr>
		<tr>
			<td>c-grid: cEEGrid</td>
			<td>TSMI, Oldenzaal, The Netherlands</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>cEEGrid connector</td>
			<td>University of Oldenburg, Germany</td>
			<td></td>
			<td>costum build</td>
		</tr>
		<tr>
			<td>EEG acquisition app: Smarting</td>
			<td>mBrainTrain, Serbia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The MathWorks, Inc., USA</td>
			<td></td>
			<td>used for data analyses and creating the figures</td>
		</tr>
		<tr>
			<td>Medical tape: Leukosilk</td>
			<td>BSN medical GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mobile EEG amplifier: Smarting MOBI</td>
			<td>mBrainTrain, Serbia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>PeakTech Pr&#252;f- und Messtechnik GmbH, Germany</td>
			<td></td>
			<td>optional device to check functionality of electrodes</td>
		</tr>
		<tr>
			<td>nEEGlace</td>
			<td>University of Oldenburg, Germany</td>
			<td></td>
			<td>costumized neckspeaker with integrated EEG amplifier (Smarting, mBrainTrain, Serbia) and cEEGrid connectors</td>
		</tr>
		<tr>
			<td>Paper wipes&#160;</td>
			<td>-</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Record-a app</td>
			<td>University of Oldenburg, Germany</td>
			<td></td>
			<td>for our exemplary data recording, open-source: https://github.com/NeuropsyOL/Pocketable-Labs</td>
		</tr>
		<tr>
			<td>Smartphone: Google Pixel 3a&#160;</td>
			<td>Google LLC, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yahama Digital Piano P-35</td>
			<td>Hamamatsu, Japan</td>
			<td></td>
			<td>for our exemplary data recording</td>
		</tr>
	</tbody>
</table>

recording brain activity with ear-electroencephalography

The c-grid (ear-electroencephalography, sold under the name cEEGrid) is an unobtrusive and comfortable electrode array that can be used for investigating brain activity after affixing around the ear. The c-grid is suitable for use outside of the laboratory for long durations, even for the whole day. Various cognitive processes can be studied using these grids, as shown by previous research, including research beyond the lab. To record high-quality ear-EEG data, careful preparation is necessary. In this protocol, we explain the steps needed for its successful implementation. First, how to test the functionality of the grid prior to a recording is shown. Second, a description is provided on how to prepare the participant and how to fit the c-grid, which is the most important step for recording high-quality data. Third, an outline is provided on how to connect the grids to an amplifier and how to check the signal quality. In this protocol, we list best practice recommendations and tips that make c-grid recordings successful. If researchers follow this protocol, they are comprehensively equipped for experimenting with the c-grid both in and beyond the lab.

When following this protocol, the impedance of each electrode is usually below 10 k&#937; or approaches this value a few minutes after placing the grid (Figure 1), indicating a good electrode-skin contact. Of note, the impedance may still improve within 2 h after fitting it.
Figure 2 illustrates different unprocessed EEG signals. Figure 2A illustrates how the data looks when no gel is used. A conductive gel is required, and the grid does not work properly without using gel. If too much gel is used, the electrodes might be bridged. The data for this scenario are shown in Figure 2B. Bridged electrodes show exactly the same signal. When the preparation and fitting are carried out carefully, one can expect high-quality data, as displayed in Figure 2C.
Figure 3 illustrates the procedure and data from an exemplary event-related potential (ERP) paradigm (oddball task) with one participant. Figure 3A illustrates the paradigm. Specifically, the experimenter played a predefined sequence of two different notes on the piano (middle C and middle G). Middle C was played frequently (328 times), and middle G was played infrequently (78 times); the participant had to count the infrequent notes. The open-source AFEx app recorded the tone onsets, loudness (RMS), and spectral content (PSD) for all the tones. The Record-A app concurrently recorded the acoustic features and EEG4. In the analyses, infrequent and frequent tones were differentiated based on the power spectral density (PSD; see H&#246;lle et al.19 for details). The EEG data were high-pass filtered at 0.1 Hz and low-pass filtered at 25 Hz. A spatial filter was computed by using generalized eigenvector decomposition, which maximizes the signal of interest20. In Figure 3B,C, the resulting ERP with typical components of auditory processing can be observed, such as the N1 for both tones and the P3 for the infrequent tone that had to be counted. These results are consistent with previous oddball studies both with c-grids1, 3 and with cap-EEG21,22.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64897/64897fig01.jpg" /> 
Figure 1: Example of good impedance. All values are in kiloohms (k&#937;). <a href="https://www.jove.com/files/ftp_upload/64897/64897fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64897/64897fig02.jpg" /> 
Figure 2: Illustration of unprocessed signals with different qualities. (A) Example of 10 s of data when no electrode gel is used at all. (B) Example of 10 s of data when the electrodes are bridged. (C) Example of 10 s of good data acquired in the lab. <a href="https://www.jove.com/files/ftp_upload/64897/64897fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64897/64897fig03.jpg" /> 
Figure 3: Results from an event-related potential (ERP) paradigm (oddball task) with one participant. (A) Overview of the paradigm. The participant listened to a sequence of tones played on a piano and had to count the infrequent one. The smartphone concurrently recorded the EEG and acoustic features (B) ERPs of all the c-grid channels. Abbreviations: REF = reference electrode; DRL = ground electrode. (C) ERP based on the spatial filter displayed in the top-left corner. <a href="https://www.jove.com/files/ftp_upload/64897/64897fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Recording Brain Activity with Ear-Electroencephalography at JoVE.com

Recording Brain Activity with Ear-Electroencephalography

Presented here is the procedure for using the c-grid (ear-electroencephalography, sold under the name cEEGrid) for recording brain activity in and beyond the lab for extended durations. This protocol describes how to set up these arrays and how to record brain activity using them.

The c-grid (ear-electroencephalography, sold under the name cEEGrid) is an unobtrusive and comfortable electrode array that can be used for investigating ...

recording-brain-activity-with-ear-electroencephalography

Research

JoVE Journal

Neuroscience

2.6K Views.  University of Oldenburg. Presented here is the procedure for using the c-grid (ear-electroencephalography, sold under the name cEEGrid) for recording brain activity in and beyond the lab for extended durations. This protocol describes how to set up these arrays and how to record brain activity using them.

Video: Recording Brain Activity with Ear-Electroencephalography

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Recording Brain Electromagnetic Activity During the Administration of the Gaseous Anesthetic Agents Xenon and Nitrous Oxide in Healthy Volunteers

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most neuroimaging or neurophysiological investigations in humans have been confined to the study of &#947;-Amino-Butyric-Acid-(GABA)-receptor-agonist-based anesthetics, while the effects of dissociative N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthetics ketamine, nitrous oxide (N2O) and xenon (Xe) are largely unknown. This paper describes the methods underlying the simultaneous recording of magnetoencephalography (MEG) and electroencephalography (EEG) from healthy males during inhalation of the gaseous anesthetic agents N2O and Xe. Combining MEG and EEG data enables the assessment of electromagnetic brain activity during anesthesia at high temporal, and moderate spatial, resolution. Here we describe a detailed protocol, refined over multiple recording sessions, that includes subject recruitment, anesthesia equipment setup in the MEG scanner room, data collection and basic data analysis. In this protocol each participant is exposed to varying levels of Xe and N2O in a repeated measures cross-over design. Following relevant baseline recordings participants are exposed to step-wise increasing inspired concentrations of Xe and N2O of 8, 16, 24 and 42%, and 16, 32 and 47% respectively, during which their level of responsiveness is tracked with an auditory continuous performance task (aCPT). Results are presented for a number of recordings to highlight the sensor-level properties of the raw data, the spectral topography, the minimization of head movements, and the unequivocal level dependent effects on the auditory evoked responses. This paradigm describes a general approach to the recording of electromagnetic signals associated with the action of different kinds of gaseous anesthetics, which can be readily adapted to be used with volatile and intravenous anesthetic agents. It is expected that the method outlined can contribute to the understanding of the macro-scale mechanisms of anesthesia by enabling methodological extensions involving source space imaging and functional network analysis.

Simultaneous magnetoencephalography and electroencephalography provides a useful tool to search for common and distinct macro-scale mechanisms of reductions in consciousness induced by different anesthetics. This paper illustrates the empirical methods underlying the recording of such data from healthy humans during N-Methyl-D-Aspartate-(NMDA)-receptor-antagonist-based anesthesia during inhalation of nitrous oxide and xenon.

Anesthesia arguably provides one of the only systematic ways to study the neural correlates of global consciousness/unconsciousness. However to date most ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Analyzing Neural Activity and Connectivity Using Intracranial EEG Data with SPM Software

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive neuroscience. Intracranial electroencephalography (EEG) can directly record electrical neural activity and has the unique potential to accomplish this goal. Traditionally, averaging analysis has been applied to analyze intracranial EEG data; however, several new techniques are available for depicting neural activity and intra- and inter-regional connectivity. Here, we introduce two analytical protocols we recently applied to analyze intracranial EEG data using the Statistical Parametric Mapping (SPM) software: time-frequency SPM analysis for neural activity and dynamic causal modeling of induced responses for intra- and inter-regional connectivity. We report our analysis of intracranial EEG data during the observation of faces as representative results. The results revealed that the inferior occipital gyrus (IOG) showed gamma-band activity at very early stages (110 ms) in response to faces, and both the IOG and amygdala showed rapid intra- and inter-regional connectivity using various types of oscillations. These analytical protocols have the potential to identify the neural mechanisms underlying cognitive functions with high spatial and temporal profiles.

We present two analytical protocols that can be used to analyze intracranial electroencephalography data using the Statistical Parametric Mapping (SPM) software: time-frequency statistical parametric mapping analysis for neural activity, and dynamic causal modeling of induced responses for intra- and inter-regional connectivity.

Measuring neural activity and connectivity associated with cognitive functions at high spatial and temporal resolutions is an important goal in cognitive ...

Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer&#39;s disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical ...