This work is supported by the ECNU Academic Innovation Promotion Program for Excellent Doctoral Students (YBNLTS2019-025) and the National Natural Science Foundation of China (31872783 and 71942001).

All recruited participants (40 dyads, mean age 22.1 &#177; 1.2 years; 100% right-handed; normal or corrected-to-normal vision) were healthy.&#160;Before the experiment, participants gave informed consent. Participants were financially compensated for their participation. The study was approved by the University Committee of Human Research Protection (HR-0053-2021), East China Normal University.
1. Preparation steps before adopting data
<ol>
	<li>Homemade NIRS caps
	<ol>
		<li>Adopt elastic swimming cap to place optode holder grid. 
		NOTE: Considering that the head sizes of the participants are different, two sizes of caps are used. Small caps are prepared for participants with a head circumference of 55.4 &#177; 1.1 cm, and large caps are for the participants with a head circumference of 57.9 &#177; 1.2 cm.</li>
		<li>Anchor the location of the EEG electrodes (inion, Cz, T3, T4, Fpz, and P5) as reference optodes according to the standard international 10-10 system on elastic swimming caps (see Table of Materials).
		<ol>
			<li>First, place the standard 10-10 EEG cap (see Table of Materials) on the head mold, and put the elastic swimming cap on the EEG cap. Second, mark reference optodes (inion, Cz, T3, T4, Fpz, and P5) with chalk on each cap. Finally, cut two holes about 15 mm in diameter to place the two reference optodes (i.e., Fpz and P5, Figure 1). 
			NOTE: Specifically, a 3 x 5 optode probe set and a 4 x 4 optode probe set are separately placed over the prefrontal area (reference optode is placed at Fpz, Figure 1B) and left temporoparietal regions (reference optode is placed at P5, Figure 1B).</li>
		</ol>
		</li>
		<li>Cut holes to place the other optodes. Arrange a swimming cap with two grid holders directly on the head mold. Then, mark the location of other optodes with chalk. After that, cut the rest holes to make sure the grid holder fits in.</li>
		<li>Mount two probe sets (i.e., 3 x 5 and 4 x 4) to the swimming caps (see Table of Materials). 
		NOTE: The NIRS measurement system (see Table of Materials) provides these standard probe sets (i.e., 3 x 5 and 4 x 4) with standard holder sockets ensuring the 30 mm optode separation.</li>
		<li>Open the probe set monitor window at the NIRS measurement system and select four probe sets arranged in 3 x 5 and 4 x 4 for each person, separately. 
		NOTE: The probe arrangements of the two caps should correspond to the structures in the probe set window (i.e., the exact location of the receiver probe numbers and the respective emitter).</li>
	</ol>
	</li>
	<li>Preparation of the experiment
	<ol>
		<li>Before recording data, ensure the NIRS system is keeping a stable operating temperature by starting the system for at least 30 min. 
		NOTE: The stable operating temperature ranged from 5 &#176;C to 35 &#176;C.</li>
		<li>Set the measurement mode to event-related measurement. Ensure the triggers receiver is active (i.e., the RS232 serial input). 
		NOTE: The experiment is programmed in commercially available psychology software (see Table of Materials). The absorption of near-infrared light (two wavelengths: 695 and 830 nm) is measured with a sampling rate of 10 Hz.</li>
		<li>Prepare the lighted fiber optic probe, which can be used to move hair aside.</li>
		<li>Set the experiment environment with one table with two chairs to keep participants&#39; seats face-to-face.</li>
	</ol>
	</li>
</ol>
2. Adopting data by instructing participants
<ol>
	<li>Prepare the participants
	<ol>
		<li>Instruct the participants, including the details of NIRS measurement methods. 
		NOTE: All the participants were healthy and were financially compensated for participation. No participants withdrew from the experiment halfway through. The laser beam of the NIRS may be harmful to the participants&#39; eyes, and they were instructed not to look directly into those laser beams.</li>
		<li>Make the participants sit face-to-face (apart from a table (0.8 m)) to make sure they can see each other directly. Adjust the chair-to-table distance (i.e., nearly 0.3 m) to make the participants sit comfortably.</li>
		<li>Turn on the laser button, and place the caps with the probe sets on the participants&#39; heads. 
		NOTE: The 3 x 5 probe sets cover the forehead of the participants (middle probe of the bottom row is placed on Fpz); the 4 x 4 probe sets cover the left temporoparietal cortex (the third probe of the third row is placed on P5).</li>
		<li>Put the four optical fiber bundles loosely on the holder&#39;s arms without contact with the participants or chairs. 
		NOTE: Here, the NIRS measurement system has four bundles of optical fibers. Additionally, ensure that the participants do not feel too heavy to pull off the caps.</li>
		<li>Let the probe tips touch participants&#39; scalp by carefully pushing each spring load probe further into its socket.</li>
		<li>Perform signal calibration.
		<ol>
			<li>First, check the quality of the signal by clicking the Auto Gain in the probe set monitor window of the fNIRS machine. Then, a channel&#39;s poor signal and sufficient signal are marked in yellow and green in the probe set monitor window, respectively. 
			NOTE: For a channel with insufficient signals, lighted fiber optic probes are used to move the hair under the probe&#39;s tip to one side.</li>
			<li>Then, push the probes further into their sockets to get sufficient signals. Repeat this process until all the channels are marked in green in the probe set monitor window of the NIRS measurement system, indicating that the quality of signals is accessible.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Run the experiment
	<ol>
		<li>Start the experiment with a 5 min rest state, which serves as the baseline. Then, two participants are required to co-learn the learning materials.</li>
		<li>After the experiment, click on Text File Out to export the raw light intensity data and save the data as a text file. 
		NOTE: No filters are applied in the NIRS measurement system.</li>
		<li>Use the three-dimensional (3D) digitizer (see Table of Materials) to determine the locations of emitters, receivers, and other references (i.e., inion, nasion, Cz, and left and right ears) for each participant.
		<ol>
			<li>Obtain the MNI coordinates for the recording channels using the commercially available numeric computing platform21 (see Table of Materials). Supplementary Table S1 shows the corresponding anatomical locations of each channel.</li>
		</ol>
		</li>
		<li>Clean probes and probe holders with ethanol. Wash caps with mild detergent and let the caps air dry.</li>
	</ol>
	</li>
</ol>
3. Data analysis
<ol>
	<li>Data preprocessing 
	NOTE: Previous research has adopted variable non-commercial software packages (e.g., Homer222, AnalyzIR23, or nirs LAB24) with numeric computing platforms (see Table of Materials) on fNIRS data analysis, and they&#160;are all available on the website. Here Homer2 was used to do the preprocessing of the NIRS data. Additionally, both fNIRS recording data collected in the rest and collaborative learning phases share the same preprocessing and analysis pipeline.
	<ol>
		<li>Copy the dataset from the fNIRS machine. Convert the original data formation to the proper formation (i.e., convert cvs file to nirs file).</li>
		<li>Convert raw data to optical density (OD) data with &#34;hmrIntensity2OD&#34; function provided in the Numeric computing platform (see Table of Materials).</li>
		<li>Delete the bad channels. Then average the OD value for each participant on each channel and full sample points, respectively. 
		NOTE: Here, 46 averaged OD values are obtained.
		<ol>
			<li>Calculate the standard deviation (SD) for each participant.</li>
			<li>Mark as unusable and remove the channels with very low or high OD (which exceeded 5 SDs) from the analysis for each participant. 
			NOTE: This step can be performed before and/or after fNIRS data preprocessing. In this data analyses pipeline, the bad channels are detected before the fNIRS data preprocessing.</li>
		</ol>
		</li>
		<li>Convert the OD time data into Oxy-Hb, DeOxy-Hb, and combined signal based on the modified Beer-Lambert Law25. 
		NOTE: Reference25 says, &#34;All data analysis steps are conducted on Oxy-Hb data, which is an indicator of the change in regional cerebral blood flow having higher signal-to-noise ratio26. Additionally, previous research employed fNIRS hyperscanning in teaching and learning scenarios mainly focused on Oxy-Hb concentration11,12,13,14.&#34;</li>
		<li>Calibrate Oxy-Hb time series from motion artifacts by the channel-by-channel wavelet-based method. 
		NOTE: Specifically, the Daubechies 5 (db5) wavelet with tuning parameter at 0.1 (see details in Homer2 manual)27,28 is adopted in removing motion artifacts.</li>
		<li>Apply the band-pass filter (i.e., 0.01-1 Hz) on the calibrated Oxy-Hb data to reduce the high-frequency noise and slow drift.</li>
		<li>Conduct principal components analysis (PCA) on the OxyHb signal to remove non-neural global components (e.g., blood pressure, respiration, and blood flow variation)29. 
		NOTE: The PCA analysis proposed by Zhang and colleagues29 is adopted here.
		<ol>
			<li>First, decompose the signal. 
			NOTE: The specific formula of decomposition of fNIRS signal is: H = U&#931;VT. Here, temporal and spatial patterns of fNIRS data are presented in two matrices (i.e., U and V). U is a 2D (sample point x principal component) matrix. V is also a 2D (principal component x principal component) matrix. The column in V indicates one principal component (PC), and the strength of that PC for a particular channel is estimated in each entry of the column. The relative importance of each PC is represented by the value of the diagonal matrix &#931;.</li>
			<li>Second, conduct spatial smoothing. 
			NOTE: Gaussian kernel convolution is employed to remove localized signals and get the global component.</li>
			<li>Third, reconstruct the signal. 
			NOTE: To calculate the global component of the fNIRS data, the smoothed spatial pattern matrix V* is plugged back into the decomposition formula: HGlobal = U&#931;(V*)T. Then, localized derived neuronal signal can be obtained using original data H to subtract HGlobal : HNeuronal = H - HGlobal.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Inter-brain synchrony 
	NOTE: To reveal brain coupling in second-person neuroscience, wavelet transform coherence (WTC) is adopted here. Briefly, WTC measures the correlation between two-time series as a function of frequency and time. The specific formula of wavelet coherence of two-time series x and y is: 
	<img alt="Equation 1" src="/files/ftp_upload/62777/62777eq01.jpg" /> 
	&#8203;T and s denote the time and wavelet scale separately, &#8249;&#183;&#8250; indicates a smoothing operation in scale and time. W represents the continuous wavelet transform. Then, a 2D (time x frequency) WTC matrix is generated30. Several toolboxes are used to calculate the WTC value. Here the toolbox created by Grinsted and colleagues was used30.
	<ol>
		<li>Adopt the WTC function of the numeric computing platform (see Table of Materials). 
		NOTE: Here, the default setting of the mother wavelet (i.e., Generalized Morse Wavelet with its parameters beta and gamma) is used. Mother wavelet converts each time series into the frequency and time domain.</li>
		<li>Set the default setting on the other parameters (i.e., MonteCarloCount, representing the number of surrogate data sets in the significance calculation).</li>
		<li>Calculate the WTC value for two corresponding channels (the same channel in two participants) in a numeric computing platform (see Table of Materials). Following the same procedure, 46 WTC matrices are generated from 46 channels.</li>
		<li>Determine the frequency band of interest (FOI), which is sensitive to collaborative learning. 
		NOTE: Here, a cluster-based permutation approach is adopted to detect such FOI31, which offers a solution to multiple comparisons in multi-channel and multi-frequency data.
		<ol>
			<li>Perform Time-average of the WTC values in the resting and collaborative learning phases, respectively, for each channel combination. Then, conduct paired sample t-tests along with the entire frequency (frequency range: 0.01-1Hz32) on these time-averaged WTC values (collaborative learning vs. rest). Next, identify frequency bins at which the task effect is significant (collaborative learning &#62; rest, p &#60; 0.05).</li>
			<li>Obtain significant frequency neighboring points (&#8805;2) as observed clusters and corresponding T values.</li>
			<li>Conduct a series of paired sample t-tests on permuted data to generate the T values for each cluster qualified in step 3.2.4.2 for 1000 times. 
			NOTE: For forming permuted data, participants are randomly assigned to form new two-member pairs. As the length of datasets varied across dyads for each random pair, the longer dataset is trimmed to the same length as the shorter one33.</li>
			<li>Compare the averaged cluster-based T values from original pairs with the T values of 1000 permutations. 
			NOTE: The p values evaluated by this formula34: 
			<img alt="Equation 2" src="/files/ftp_upload/62777/62777eq02.jpg" />&#160;, where S0 denotes observed averaged-cluster t-value, &#956;p and &#963;p indicate the mean and standard deviation of permutation values.</li>
			<li>Average WTC values in the identified FOI in each channel in each dyad. Then, apply fisher z transformation to the WTC values to get a normal distribution of WTC values. Use this value to index the IBS for further statistical analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

First, in the present protocol, the specific steps of conducting fNIRS hyperscanning experiments in a collaborative learning scenario are stated. Second, the data analysis pipeline that assesses the IBS of hemodynamic signals in collaborative learning dyads is also presented. The detailed operation on conducting fNIRS hyperscanning experiments would promote the development of open-science. Furthermore, the analysis pipeline is provided here to increase the reproducibility of hyperscanning research. In the following, the ...

Recently, to reveal the concurrent brain activity across the interactive dyads or members of a group, researchers employ the hyperscanning approach1,2. Specifically, electroencephalogram (EEG), functional magnetic resonance imaging (fMRI), and functional near-infrared spectroscopy (fNIRS) are used to record the neural and brain activities from two or more subjects simultaneously3,4,5. Researchers extract a neural index entailing concurrent brain coupling based on this technique, which refers to inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A large variety of hyperscanning research found IBS during social interaction between multiple individuals (e.g., player-audience, instructor-learner, and leader-follower)6,7,8. Furthermore, IBS holds specific implications of effective learning and instruction9,10,11,12,13,14. With the surging of hyperscanning research in naturalistic learning scenarios, establishing a standard protocol of hyperscanning experiments and the pipeline of data analysis in this field is necessary.
Thus, this paper provides a protocol for conducting fNIRS-based hyperscanning of collaborative learning dyads and a pipeline for analyzing IBS. fNIRS is an optical imaging tool, which radiates near-infrared light to assess the spectral absorption of hemoglobin indirectly, and then hemodynamic/oxygenation activity is measured15,16,17. Compared with fMRI, fNIRS is less prone to motion artifacts, allowing measurements from subjects who are doing real-life experiments (e.g., imitation, talking, and non-verbal communication)18,7,19. In comparison with EEG, fNIRS holds higher spatial resolution, allowing researchers to detect the location of brain activity20. Thus, these advantages in spatial resolution, logistics, and feasibility qualify fNIRS to conduct hyperscanning measurement1. Using this technology, an emerging research body detects an index term as IBS-the neural alignment of two (or more) people&#39;s brain activity-in different forms of naturalistic social settings9,10,11,12,13,14. In those studies, various methods (i.e., Correlation analysis and Wavelet Transform Coherence (WTC) analysis) are applied to calculate this index; meanwhile, a standard pipeline on such analysis is essential but lacking. As a result, a protocol for conducting fNIRS-based hyperscanning and a pipeline using WTC analysis to identify IBS is presented in this work
This study aims to evaluate IBS in collaborative learning dyads using the fNIRS hyperscanning technique. First, a hemodynamic response is recorded simultaneously in each dyads&#39; prefrontal and left temporoparietal regions during a collaborative learning task. These regions have been identified as associated with interactive teaching and learning9,10,11,12,13,14. Second, the IBS is calculated on each corresponding channel. The fNIRS data recording process consists of two parts: resting-state session and collaborative session. The resting-state session lasts for 5 min, during which both the participants (sitting face-to-face, apart from one another by a table (0.8 m)) are required to remain still and relax. This resting-state session is served as the baseline. Then, in the collaborative session, the participants are told to study the entire learning materials together, eliciting understanding, summarizing the rules, and making sure all learning materials are mastered. Here, the specific steps of conducting the experiment and fNIRS data analysis are presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>EEG caps</td>
			<td>Compumedics Neuroscan,Charlotte,USA</td>
			<td>64-channel Quik-Cap</td>
			<td>We choose two sizes of cap(i.e.medium and large).</td>
		</tr>
		<tr>
			<td>NIRS measurement system with probe sets and probe holder grids</td>
			<td>Hitachi Medical Corporation, Tokyo, Japan</td>
			<td>ETG-7100 Optical Topography System</td>
			<td>The current study protocol requires an optional second adult probe set for 92 channels of measurement in total.</td>
		</tr>
		<tr>
			<td>Numeric computing platform</td>
			<td>The MathWorks, Inc., Natick, MA</td>
			<td>MATLAB R2020a</td>
			<td>Serves as base for Psychophysics Toolbox extensions (stimulus presentation), Homer2&#160; (fNIRS preprocess analysis), and &#34;wtc&#34; function(WTC computation).</td>
		</tr>
		<tr>
			<td>Psychology software</td>
			<td>psychology software tools,Sharpsburg, PA,USA</td>
			<td>E-prime 2.0</td>
			<td>we apply E-prime to start the fNIRS measurement system and send triggers which marking the rest phase and collaborative learning phase for fNIRS recording data</td>
		</tr>
		<tr>
			<td>Swimming caps</td>
			<td>Zoke corporation,Shanghai,China</td>
			<td>611503314</td>
			<td>We first placed the standard 10-20 EEG cap on the head mold, and placed the swimming cap on the EEG cap. Second, we marked (inion, Cz, T3, T4, PFC and P5) with chalk.</td>
		</tr>
		<tr>
			<td>Three-dimensional (3-D) digitizer</td>
			<td>Polhemus, Colchester, VT, USA;</td>
			<td>Three-dimensional (3-D) digitizer</td>
			<td>Anatomical locations of optodes in relation to standard head landmarks were determined for each participant using a Patriot 3D Digitizer</td>
		</tr>
	</tbody>
</table>

inter-brain synchrony in open-ended collaborative learning: an fnirs-hyperscanning study

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the concurrent brain activity of two or more interactive individuals with a novel index called inter-brain synchrony (IBS) (i.e., phase and/or amplitude alignment of the neuronal or hemodynamic signals across time). A protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is presented here. Further, a pipeline of analyzing IBS of oxygenated hemoglobin (Oxy-Hb) signal is explained. Specifically, the experimental design, the process of NIRS data recording, data analysis methods, and future directions are all discussed. Overall, implementing a standardized fNIRS hyperscanning pipeline is a fundamental part of second-person neuroscience. Also, this is in line with the call for open-science to aid the reproducibility of research.

Figure 1 illustrates the experimental protocol and probe location. The fNIRS data recording process consists of two parts: resting-state session (5 min) and collaborative session (15-20 min). The collaborative learning dyads are required to relax and to keep still in the resting-state session. After that, participants are told to co-learning the learning material (Figure 1A). Their prefrontal and left temporoparietal regions are covered by the corresponding prob...

Watch this Scientific Journal Video about Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study at JoVE.com

Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

fNIRS hyperscanning is widely used to detect the neurobiological underpinnings of social interaction. With this technique, researchers qualify the ...

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4.0K Views.  East China Normal University. The protocol for conducting fNIRS hyperscanning experiments on collaborative learning dyads in a naturalistic learning environment is outlined. Further, a pipeline to analyze the Inter-Brain Synchrony (IBS) of oxygenated hemoglobin (Oxy-Hb) signals is presented.

Video: Inter-Brain Synchrony in Open-Ended Collaborative Learning: An fNIRS-Hyperscanning Study

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Mapping Cortical Dynamics Using Simultaneous MEG/EEG and Anatomically-constrained Minimum-norm Estimates: an Auditory Attention Example

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the cortical networks involved in dynamical perceptual and cognitive tasks, such as attending to different sounds in a cocktail party. Many past studies have employed data recorded at the sensor level only, i.e., the magnetic fields or the electric potentials recorded outside and on the scalp, and have usually focused on activity that is time-locked to the stimulus presentation. This type of event-related field / potential analysis is particularly useful when there are only a small number of distinct dipolar patterns that can be isolated and identified in space and time. Alternatively, by utilizing anatomical information, these distinct field patterns can be localized as current sources on the cortex. However, for a more sustained response that may not be time-locked to a specific stimulus (e.g., in preparation for listening to one of the two simultaneously presented spoken digits based on the cued auditory feature) or may be distributed across multiple spatial locations unknown a priori, the recruitment of a distributed cortical network may not be adequately captured by using a limited number of focal sources.
Here, we describe a procedure that employs individual anatomical MRI data to establish a relationship between the sensor information and the dipole activation on the cortex through the use of minimum-norm estimates (MNE). This inverse imaging approach provides us a tool for distributed source analysis. For illustrative purposes, we will describe all procedures using FreeSurfer and MNE software, both freely available. We will summarize the MRI sequences and analysis steps required to produce a forward model that enables us to relate the expected field pattern caused by the dipoles distributed on the cortex onto the M/EEG sensors. Next, we will step through the necessary processes that facilitate us in denoising the sensor data from environmental and physiological contaminants. We will then outline the procedure for combining and mapping MEG/EEG sensor data onto the cortical space, thereby producing a family of time-series of cortical dipole activation on the brain surface (or "brain movies") related to each experimental condition. Finally, we will highlight a few statistical techniques that enable us to make scientific inference across a subject population (i.e., perform group-level analysis) based on a common cortical coordinate space.

We use magneto- and electroencephalography (MEG/EEG), combined with anatomical information captured by magnetic resonance imaging (MRI), to map the dynamics of the cortical network associated with auditory attention.

Magneto- and electroencephalography (MEG/EEG) are neuroimaging techniques that provide a high temporal resolution particularly suitable to investigate the ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Microstate and Omega Complexity Analyses of the Resting-state Electroencephalography

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of EEG data and have been widely used to investigate the neural mechanisms in some brain disorders. The goal of this article is to describe the protocol underlying EEG microstate and omega complexity analyses step by step. The main advantage of these two measures is that they could eliminate the reference-dependent problem inherent to traditional spectrum analysis. In addition, microstate analysis makes good use of high time resolution of resting-state EEG, and the four obtained microstate classes could match the corresponding resting-state networks respectively. The omega complexity characterizes the spatial complexity of the whole brain or specific brain regions, which has obvious advantage compared with traditional complexity measures focusing on the signal complexity in a single channel. These two EEG measures could complement each other to investigate the brain complexity from the temporal and spatial domain respectively.

This article describes the protocol underlying electroencephalography (EEG) microstate analysis and omega complexity analysis, which are two reference-free EEG measures and highly valuable to explore the neural mechanisms of brain disorders.

Microstate and omega complexity are two reference-free electroencephalography (EEG) measures that can represent the temporal and spatial complexities of ...

An Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance imaging (fMRI) studies indicates that the anterior cingulate (ACC) and the lateral prefrontal cortices (latPFC) are essential nodes subserving cognitive control. However, because of its limited temporal resolution, fMRI cannot accurately reflect the timing and nature of their presumed interplay. The present study combines distributed source modeling of the temporally precise magnetoencephalography (MEG) signal with structural MRI in the form of &#34;brain movies&#34; to: (1) estimate the cortical areas involved in cognitive control (&#34;where&#34;), (2) characterize their temporal sequence (&#34;when&#34;), and (3) quantify the oscillatory dynamics of their neural interactions in real time. Stroop interference was associated with greater event-related theta (4 - 7 Hz) power in the ACC during conflict detection followed by sustained sensitivity to cognitive demands in the ACC and latPFC during integration and response preparation. A phase-locking analysis revealed co-oscillatory interactions between these areas indicating their increased neural synchrony in theta band during conflict-inducing incongruous trials. These results confirm that theta oscillations are fundamental to long-range synchronization needed for integrating top-down influences during cognitive control. MEG reflects neural activity directly, which makes it suitable for pharmacological manipulations in contrast to fMRI that is sensitive to vasoactive confounds. In the present study, healthy social drinkers were given a moderate alcohol dose and placebo in a within-subject design. Acute intoxication attenuated theta power to Stroop conflict and dysregulated co-oscillations between the ACC and latPFC, confirming that alcohol is detrimental to neural synchrony subserving cognitive control. It interferes with goal-directed behavior that may result in deficient self-control, contributing to compulsive drinking. In sum, this method can provide insight into real-time interactions during cognitive processing and can characterize the selective sensitivity to pharmacological challenge across relevant neural networks.

This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance ...

Shuttle Box Assay as an Associative Learning Tool for Cognitive Assessment in Learning and Memory Studies using Adult Zebrafish

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and traumatic brain injury (TBI). Zebrafish are an important neuroscience model due to their transparency during development and robust regenerative capabilities following neurotrauma. While various cognitive tests exist in zebrafish, most of the cognitive assessments that are rapid examine non-associative learning. At the same time, associative-learning assays often require multiple days or weeks. Here, we describe a rapid associative-learning test that utilizes an adverse stimulus (electric shock) and requires minimal preparation time. The shuttle box assay, presented here, is simple, ideal for novice investigators, and requires minimal equipment. We demonstrate that, following TBI, this shuttle box test reproducibly assesses cognitive deficit and recovery from young to old zebrafish. Additionally, the assay is adaptable to examine either immediate or delayed memory. We demonstrate that both a single TBI and repeated TBI events negatively affect learning and immediate memory but not delayed memory. We, therefore, conclude that the shuttle box assay reproducibly tracks the progression and recovery of cognitive impairment.

Learning and memory are potent metrics in studying either developmental, disease-dependent, or environmentally induced cognitive impairments. Most cognitive assessments require specialized equipment and extensive time commitments. However, the shuttle box assay is an associative learning tool that utilizes a conventional gel box for rapid and reliable assessment of adult zebrafish cognition.

Cognitive deficits, including impaired learning and memory, are a primary symptom of various developmental and age-related neurodegenerative diseases and ...

How to Calculate and Validate Inter-brain Synchronization in a fNIRS Hyperscanning Study

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each other, mostly using simultaneous-recording signals of brains (namely hyperscanning) with fNIRS. In fNIRS hyperscanning studies, IBS has been commonly assessed through the wavelet transform coherence (WTC) method because of its advantage on expanding time series into time-frequency space where oscillations can be seen in a highly intuitive way. The observed IBS can be further validated via the permutation-based random pairing of the trial, partner, and condition. Here, a protocol is presented to describe how to obtain brain signals via fNIRS technology, calculate IBS through the WTC method, and validate IBS by permutation in a hyperscanning study. Further, we discuss the critical issues when using the above methods, including the choice of fNIRS signals, methods of data preprocessing, and optional parameters of computations.&#160;In summary, using the WTC method and permutation is a potentially standard pipeline for analyzing IBS in fNIRS hyperscanning studies, contributing to both the reproducibility and reliability of IBS.

The dynamics between coupled brains of individuals have been increasingly represented by inter-brain synchronization (IBS) when they coordinate with each ...

Measurement of the Directional Information Flow in fNIRS-Hyperscanning Data using the Partial Wavelet Transform Coherence Method

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural synchronization (INS) during social interactions, functional near-infrared spectroscopy (fNIRS) is one of the most popular techniques for hyperscanning naturalistic social interactions because of its relatively high spatial resolution, sound anatomical localization, and exceptionally high tolerance of motion artifacts. Previous fNIRS-based hyperscanning studies usually calculate a time-lagged INS using wavelet transform coherence (WTC) to describe the direction and temporal pattern of information flow between individuals. However, the results of this method might be confounded by the autocorrelation effect of the fNIRS signal of each individual. For addressing this issue, a method termed partial wavelet transform coherence (pWTC) was introduced, which aimed to remove the autocorrelation effect and maintain the high temporal-spectrum resolution of the fNIRS signal. In this study, a simulation experiment was performed first to show the effectiveness of the pWTC in removing the impact of autocorrelation on INS. Then, step-by-step guidance was offered on the operation of the pWTC based on the fNIRS dataset from a social interaction experiment. Additionally, a comparison between the pWTC method and the traditional WTC method and that between the pWTC method and the Granger causality (GC) method was drawn. The results showed that pWTC could be used to determine the INS difference between different experimental conditions and INS's directional and temporal pattern between individuals during naturalistic social interactions. Moreover, it provides better temporal and frequency resolution than the traditional WTC and better flexibility than the GC method. Thus, pWTC is a strong candidate for inferring the direction and temporal pattern of information flow between individuals during naturalistic social interactions.

This protocol describes partial wavelet transform coherence (pWTC) for calculating the time-lagged pattern of interpersonal neural synchronization (INS) to infer the direction and temporal pattern of information flow during social interaction. The effectiveness of pWTC in removing the confounds of signal autocorrelation on INS was proved by two experiments.

Social interaction is of vital importance for human beings. While the hyperscanning approach has been extensively used to study interpersonal neural ...

Group Synchronization During Collaborative Drawing Using Functional Near-Infrared Spectroscopy

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, which is relevant for studying group interpersonal interactions in ecological settings. Although many fNIRS systems technically offer the possibility to monitor more than two individuals simultaneously, establishing easy-to-implement setup procedures and reliable paradigms to track hemodynamic and behavioral responses in group interaction is still required. The present protocol combines fNIRS and video-based observation to measure interpersonal synchronization in quartets during a cooperative task.This protocol provides practical recommendations for data acquisition and paradigm design, as well as guiding principles for an illustrative data analysis example. The procedure is designed to assess differences in brain and behavior interpersonal responses between social and non-social conditions inspired by a well-known ice-breaker activity, the Collaborative Face Drawing Task. The described procedures can guide future studies to adapt group naturalistic social interaction activities to the fNIRS environment.

The present protocol combines functional near-infrared spectroscopy (fNIRS) and video-based observationto measure interpersonal synchronization in quartets during a collaborative drawing task.

Functional near-infrared spectroscopy (fNIRS) is a noninvasive method particularly suitable for measuring cerebral cortex activation in multiple subjects, ...