This work was performed in part at the high performance computing cluster (HPCC), which is supported by information and communication technology office (ICTO) of the University of Macau. This study was supported by MYRG2019-00082-FHS and MYRG 2018-00081-FHS grants from the University of Macau in Macau, and also funded by The Science and Technology Development Fund, Macau SAR (FDCT 0011/2018/A1 and FDCT 025/2015/A1).

Prior to the experimental tests, all participants signed informed consent documents. The protocol for the present study was approved by the Ethics Committee of the University of Macau.
1. Hardware and software setting for concurrent EEG and fNIRS recordings
<ol>
	<li>Construct a head cap for concurrent EEG-fNIRS recordings.
	<ol>
		<li>Select the appropriate cap size according to the head circumference of participants. In this study, use a medium-size cap since it is suitable for most adolescent and adult participants.</li>
		<li>Design the layout of fNIRS optodes along with the EEG cap in the prefrontal cortex (Figure 1).
		<ol>
			<li>Place EEG electrodes in the middle section of the fNIRS optodes to ensure the measurement of the same brain region by the two techniques19,30. However, due to the low spatial resolution of both EEG and fNIRS neuroimaging methods, place the electrodes in the corresponding brain area covered by fNIRS optodes rather than the exact locations of fNIRS channels.</li>
			<li>Make 22 holes inside the EEG cap to hold the fNIRS optodes in line with the specific layout in the prefrontal cortex. Identify and mark the locations of fNIRS optodes according to the designed layout of the head cap and then punch holes inside the cap to place and fix the optodes.</li>
			<li>Place 21 or 71 EEG electrodes along the surface of the EEG cap (see&#160;Table of Materials) according to the 10-20 International System and mount the grids for the optodes.</li>
		</ol>
		</li>
		<li>Set the distance between each source-detector pair as 3 cm and then fix the optodes, in which the blue optodes denote the light detectors while the red ones represent the laser sources.</li>
	</ol>
	</li>
	<li>Set the EEG and fNIRS ports in the software.</li>
	<li>Use the time triggers generated through the parallel port and serial port to ensure the synchronization of two different signals.
	<ol>
		<li>Set the parallel port (e.g., H378 in this study) for the EEG system (see&#160;Table of Materials).</li>
		<li>Set the serial port (e.g., 6 9600 in this study) for the fNIRS system (see&#160;Table of Materials). 
		NOTE: The port type and number should be modified regarding various EEG and fNIRS setups. Please contact the manufacturers for more information.</li>
	</ol>
	</li>
</ol>
2. Experimental preparation
<ol>
	<li>Warm up the fNIRS system with lasers switched on for 30 min.</li>
	<li>Set all necessary operation parameters for the fNIRS measurement system.</li>
	<li>Show the fused experimental setup including the EEG and fNIRS measurement systems to participants.</li>
	<li>Measure and mark the Cz point according to the 10-20 International System. Identify the electrode position of Cz at half of the distance between the inion and nasion and half of the distance between the left and right inter-aural indentations.</li>
	<li>Place the front part of the cap along the participant&#8217;s forehead first and then pull down the back section of the cap towards the neck.</li>
	<li>Validate the positions.
	<ol>
		<li>Measure the distance between the Cz and inion and nasion again with a soft ruler, and double-check whether it is located at the midpoint. Likewise, measure the distance between the Cz and left and right inter-aural, and double-check whether the Cz is located at the midpoint.</li>
	</ol>
	</li>
	<li>Prepare for the EEG recordings. 
	NOTE: It is highly recommended that the EEG electrodes be set up first and then the fNIRS optodes. If EEG conductive gel covers the holes for the placement of fNIRS optodes, it should be cleaned to prevent the contamination of optodes.
	<ol>
		<li>Fill conductive gel by inserting a blunt needle through the holes of the EEG electrode grid.</li>
		<li>Place all electrodes into the EEG electrode grid according to the labels.</li>
		<li>Open the EEG software and inspect the signal quality of EEG electrodes.</li>
		<li>Readjust the electrode by refilling conductive gel if the signal quality is not good enough to meet the requirements (40 mV).</li>
		<li>Readjust the electrode by refilling conductive gel if the impedance could not meet the requirements.</li>
	</ol>
	</li>
	<li>Prepare for the fNIRS recordings. 
	Caution: Do not expose participants&#8217; eyes to the laser beam of fNIRS sources directly.
	<ol>
		<li>Place the optical fibers along the holder arms attached to the fNIRS measurement system as well as the holder. Ensure that the fibers are neat and tidy.</li>
		<li>Insert the optical sources and detectors into the holes according to the layout.</li>
		<li>Test the signal quality. If a channel does not have a high-level signal-to-noise ratio (i.e., if the channel is marked in yellow), gently inspect the participant&#8217;s hair surrounding the optical probes to ensure that nothing exists between the optical probe and scalp.</li>
		<li>If step 2.8.3 cannot improve the signal quality, turn up the signal intensity. If there is too much signal (i.e., if the channel is marked in red), turn down the signal intensity.</li>
	</ol>
	</li>
</ol>
3. Run the experiment
<ol>
	<li>Start the experiment when the signals are stable with excellent signal-to-noise ratio and participants are familiar with the experiment instructions. Use the classic Flanker paradigm for the experimental test29,31.</li>
	<li>After the experiment, save and export the data from both EEG and fNIRS.</li>
	<li>Remove EEG electrodes and fNIRS optical probes carefully.</li>
</ol>
4. Measurement of three-dimensional (3D) MNI coordinates of fNIRS optodes with 3D digitizer
<ol>
	<li>Let participants sit in a chair and wear the glasses with the sensor.</li>
	<li>Open the digitizer software on the computer. Ensure that the 3D digitizer system is in connection with the computer through an appropriate COM port.</li>
	<li>Load the layout of the optodes setting file.</li>
	<li>Move the 3D digitizer stylus across the key positions (Nz, Iz, left ear, right ear, Cz) along with the screen and press the button on the stylus.</li>
	<li>Localize the optical sources and detectors</li>
	<li>Export the 3D coordinates files.</li>
</ol>
5. Data analysis
<ol>
	<li>fNIRS data analysis
	<ol>
		<li>Process the 3D MNI coordinates data by using the registration option in NIRS-SPM with MATLAB 2019. Select: stand-alone spatial registration | With 3D Digitize. Choose the previously saved others and origin text files and then select Registration.</li>
		<li>Pre-process fNIRS signals with Homer2 software32.
		<ol>
			<li>Convert the raw data to optical density changes for different wavelengths and further convert to the concentration changes of HbO at different time points using a modified Beer-Lambert Law. Generally, the typically differential path length factor (DPF) value affected by the age, gender, and wavelength, and the distance between the source and decetor33,34 is 6, which is similar to the average&#160;DPF from previous studies34,35.</li>
			<li>Use the spline motion artifacts detection algorithm from the Homer2 fNIRS processing package for motion correction. Please select the appropriate methods of motion correction based on literature36.</li>
			<li>Process the raw hemoglobin continuous data by a low-pass filter of 0.2 Hz and subsequently a high-pass filter of 0.015 Hz.</li>
			<li>Normalize hemodynamic signal amplitude by dividing the averaged values.</li>
			<li>Generate the fNIRS data for each channel based on the 3D digitizer information. Select the channels that have a registration probability of 100% or more in the superior frontal cortex (SFC)according to the regression calculation of the NIRS-SPM for further analysis.</li>
			<li>Export the peak values of oxygen hemoglobin (HbO) concentration changes. 
			NOTE: In this study, only HbO signals were analyzed due to their high signal-to-noise ratio. The peak values of run-averaged HbO data were extracted for each channel from each participant for further analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>EEG data processing 
	NOTE: Offline EEG data analysis was performed with the EEGLAB. Only N200 at Fz was the interesting component for the present study. All electrodes were subjected to an automatic artifact correction to remove eye movements by using an internal model of artifact topographies. Continuous EEG data were then segmented into different trials according to target and nontarget stimuli, in which the epoch for each trial lasted 2500 ms, involving a pre-stimulus period of 500 ms (baseline epoch) and a post-stimulus period of 2000 ms (task epoch).
	<ol>
		<li>Load the raw EEG data folder into the EEGLAB by using the plugins. Choose the BIOSIG plugin for the BDF file in this study. 
		NOTE: Please choose a suitable plugin according to the EEG data file format.</li>
		<li>Set the channel location information for EEGLAB37. Load the corresponding location file of the cap.</li>
		<li>Re-reference electrodes in the ERPLAB, which is one plugin of EEGLAB. Choose the channels placed in the mastoids as reference electrodes.</li>
		<li>Extract EEG data epochs based on the event and bin files in the ERPLAB37.</li>
		<li>Filter the EEG data segments in the ERPLAB by using the FIR filter by filtering the low frequencies with a cutoff of 30Hz and by filtering the high frequencies with a cutoff of 0.1 Hz.</li>
		<li>Remove ocular EEG artifacts with the Independent Component Analysis in EEGLAB.</li>
		<li>Reject EEG data segments with amplitude values exceeding &#177; 100 &#181;V at any channel in ERPLAB.</li>
		<li>Average the EEG data segments in ERPLAB. 
		NOTE: These are the generally used data analysis method and the software for processing EEG and the fNIRS data. There are numerous processing software and methods available.</li>
	</ol>
	</li>
	<li>Correlation calculation
	<ol>
		<li>Generate the relationship between fNIRS and EEG recordings by using Pearson correlation analysis.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kennan, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+recording+of+event-related+auditory+oddball+response+using+transcranial+near+infrared+optical+topography+and+surface+EEG.">Simultaneous recording of event-related auditory oddball response using transcranial near infrared optical topography and surface EEG.</a> NeuroImage. 16 (3), 587-592 (2002).</li><li>Horovitz, S. G., Gore, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+event-related+potential+and+near-infrared+spectroscopic+studies+of+semantic+processing.">Simultaneous event-related potential and near-infrared spectroscopic studies of semantic processing.</a> Human Brain Mapping. 22 (2), 110-115 (2004).</li><li>Yuan, Z., Ye, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+of+fNIRS+and+fMRI+data:+identifying+when+and+where+hemodynamic+signals+are+changing+in+human+brains.">Fusion of fNIRS and fMRI data: identifying when and where hemodynamic signals are changing in human brains.</a> Frontiers in Human Neuroscience. 7, 676 (2013).</li><li>Lin, X., Sai, L., Yuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Concealed+Information+with+Fused+Electroencephalography+and+Functional+Near-infrared+Spectroscopy.">Detecting Concealed Information with Fused Electroencephalography and Functional Near-infrared Spectroscopy.</a> Neuroscience. 386, 284-294 (2018).</li><li>Ieong, H. F., Yuan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emotion+recognition+and+its+relation+to+prefrontal+function+and+network+in+heroin+plus+nicotine+dependence:+a+pilot+study.">Emotion recognition and its relation to prefrontal function and network in heroin plus nicotine dependence: a pilot study.</a> Neurophotonics. 5 (02), 1 (2018).</li><li>Hu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+Mapping+of+Brain+Activation+and+Connectivity+in+Occipitotemporal+Cortex+During+Chinese+Character+Recognition.">Optical Mapping of Brain Activation and Connectivity in Occipitotemporal Cortex During Chinese Character Recognition.</a> Brain Topography. 31 (6), 1014-1028 (2018).</li><li>Wang, M. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+mapping+of+brain+activation+from+multiple+subjects+during+social+interaction+by+hyperscanning:+a+mini-review.">Concurrent mapping of brain activation from multiple subjects during social interaction by hyperscanning: a mini-review.</a> Quantitative Imaging in Medicine and Surgery. 8 (8), 819-837 (2018).</li><li>Scholkmann, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+continuous+wave+functional+near-infrared+spectroscopy+and+imaging+instrumentation+and+methodology.">A review on continuous wave functional near-infrared spectroscopy and imaging instrumentation and methodology.</a> NeuroImage. 85, 6-27 (2014).</li><li>Wan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+basis+of+the+hemodynamic+response+nonlinearity+in+human+primary+visual+cortex:+Implications+for+neurovascular+coupling+mechanism.">The neural basis of the hemodynamic response nonlinearity in human primary visual cortex: Implications for neurovascular coupling mechanism.</a> NeuroImage. 32 (2), 616-625 (2006).</li><li>Miller, E. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prefontral+cortex+and+cognitive+control.">The prefontral cortex and cognitive control.</a> Nature Reviews Neuroscience. 1 (1), 59-65 (2000).</li><li>Miller, E. K., Cohen, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrative+theory+of+prefrontal+cortex+function.">An integrative theory of prefrontal cortex function.</a> Annual review of Neuroscience. 24 (1), 167-202 (2001).</li><li>Mansouri, F. A., Tanaka, K., Buckley, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conflict-induced+behavioural+adjustment:+a+clue+to+the+executive+functions+of+the+prefrontal+cortex.">Conflict-induced behavioural adjustment: a clue to the executive functions of the prefrontal cortex.</a> Nature Reviews Neuroscience. 10 (2), 141-152 (2009).</li><li>Wood, J. N., Grafman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+prefrontal+cortex:+processing+and+representational+perspectives.">Human prefrontal cortex: processing and representational perspectives.</a> Nature Reviews Neuroscience. 4 (2), 139-147 (2003).</li><li>Wallis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orbitofrontal+Cortex+and+Its+Contribution+to+Decision-Making.">Orbitofrontal Cortex and Its Contribution to Decision-Making.</a> Annual Review of Neuroscience. 30 (1), 31-56 (2007).</li><li>Forbes, C. E., Grafman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+the+Human+Prefrontal+Cortex+in+Social+Cognition+and+Moral+Judgment.">The Role of the Human Prefrontal Cortex in Social Cognition and Moral Judgment.</a> Annual Review of Neuroscience. 33 (1), 299-324 (2010).</li><li>Nguyen, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+continuous+EEG-fNIRS+recording+of+temporal+lobe+seizures.">Non-invasive continuous EEG-fNIRS recording of temporal lobe seizures.</a> Epilepsy Research. 99 (1-2), 112-126 (2012).</li><li>Peng, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fNIRS-EEG+study+of+focal+interictal+epileptiform+discharges.">fNIRS-EEG study of focal interictal epileptiform discharges.</a> Epilepsy Research. 108 (3), 491-505 (2014).</li><li>Liu, Y., Ayaz, H., Shewokis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multisubject+"learning"+for+mental+workload+classification+using+concurrent+EEG,+fNIRS,+and+physiological+measures.">Multisubject "learning" for mental workload classification using concurrent EEG, fNIRS, and physiological measures.</a> Frontiers in Human Neuroscience. 11, (2017).</li><li>Aghajani, H., Garbey, M., Omurtag, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+mental+workload+with+EEG+fNIRS.">Measuring mental workload with EEG+fNIRS.</a> Frontiers in Human Neuroscience. 11, (2017).</li><li>Balconi, M., Vanutelli, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+(fNIRS)+and+EEG+(N200)+correlates+of+emotional+inter-species+interactions+modulated+by+visual+and+auditory+stimulation.">Hemodynamic (fNIRS) and EEG (N200) correlates of emotional inter-species interactions modulated by visual and auditory stimulation.</a> Scientific Reports. 6, (2016).</li><li>Donohue, S. E., Appelbaum, L. G., McKay, C. C., Woldorff, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neural+dynamics+of+stimulus+and+response+conflict+processing+as+a+function+of+response+complexity+and+task+demands.">The neural dynamics of stimulus and response conflict processing as a function of response complexity and task demands.</a> Neuropsychologia. 84, 14-28 (2016).</li><li>Liu, Y., Ayaz, H., Shewokis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mental+workload+classification+with+concurrent+electroencephalography+and+functional+near-infrared+spectroscopy.">Mental workload classification with concurrent electroencephalography and functional near-infrared spectroscopy.</a> Brain-Computer Interfaces. 4 (3), 175-185 (2017).</li><li>Fazli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+performance+by+a+hybrid+NIRS-EEG+brain+computer+interface.">Enhanced performance by a hybrid NIRS-EEG brain computer interface.</a> NeuroImage. 59 (1), 519-529 (2012).</li><li>Putze, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+fNIRS-EEG+based+classification+of+auditory+and+visual+perception+processes.">Hybrid fNIRS-EEG based classification of auditory and visual perception processes.</a> Frontiers in Neuroscience. 8, 373 (2014).</li><li>Horovitz, S. G., Gore, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+event-related+potential+and+near-infrared+spectroscopic+studies+of+semantic+processing.">Simultaneous event-related potential and near-infrared spectroscopic studies of semantic processing.</a> Human Brain Mapping. 22 (2), 110-115 (2004).</li><li>Lin, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+small-world+properties+of+brain+networks+in+Chinese+to+English+simultaneous+interpreting+by+using+functional+near-infrared+spectroscopy.">Mapping the small-world properties of brain networks in Chinese to English simultaneous interpreting by using functional near-infrared spectroscopy.</a> Journal of Innovative Optical Health Sciences. 11 (03), 1840001 (2018).</li><li>Folstein, J. R., Van Petten, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cognitive+control+and+mismatch+on+the+N2+component+of+the+ERP:+A+review.">Influence of cognitive control and mismatch on the N2 component of the ERP: A review.</a> Psychophysiology. 45 (1), 152 (2008).</li><li>Patel, S. H., Azzam, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+N200+and+P300:+Selected+studies+of+the+Event-Related+Potential.">Characterization of N200 and P300: Selected studies of the Event-Related Potential.</a> International Journal of Medical Sciences. 2 (4), 147-154 (2005).</li><li>Suzuki, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+the+superior+frontal+cortex+and+alpha+oscillation+in+a+flanker+task:+Simultaneous+recording+of+electroencephalogram+(EEG)+and+near+infrared+spectroscopy+(NIRS).">The relationship between the superior frontal cortex and alpha oscillation in a flanker task: Simultaneous recording of electroencephalogram (EEG) and near infrared spectroscopy (NIRS).</a> Neuroscience Research. 131, 30-35 (2018).</li><li>Keles, H. O., Barbour, R. L., Omurtag, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+correlates+of+spontaneous+neural+activity+measured+by+human+whole-head+resting+state+EEG+++fNIRS.">Hemodynamic correlates of spontaneous neural activity measured by human whole-head resting state EEG + fNIRS.</a> NeuroImage. 138, 76-87 (2016).</li><li>Eriksen, B. A., Eriksen, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+noise+letters+upon+the+identification+of+a+target+letter+in+a+nonsearch+task.">Effects of noise letters upon the identification of a target letter in a nonsearch task.</a> Perception &amp; Psychophysics. 16 (1), 143-149 (1974).</li><li>Huppert, T. J., Diamond, S. G., Franceschini, M. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HomER:+a+review+of+time-series+analysis+methods+for+near-infrared+spectroscopy+of+the+brain.">HomER: a review of time-series analysis methods for near-infrared spectroscopy of the brain.</a> Applied optics. 48 (10), 280-289 (2009).</li><li>Kocsis, L., Herman, P., Eke, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+modified+Beer-Lambert+law+revisited.">The modified Beer-Lambert law revisited.</a> Physics in Medicine and Biology. 51 (5), (2006).</li><li>Herold, F., Wiegel, P., Scholkmann, F., M&#252;ller, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Functional+Near-Infrared+Spectroscopy+(fNIRS)+Neuroimaging+in+Exercise-Cognition+Science:+A+Systematic,+Methodology-Focused+Review.">Applications of Functional Near-Infrared Spectroscopy (fNIRS) Neuroimaging in Exercise-Cognition Science: A Systematic, Methodology-Focused Review.</a> Journal of Clinical Medicine. 7 (12), 466 (2018).</li><li>Duncan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+pathlength+measurements+on+adult+head,+calf+and+forearm+and+the+head+of+the+newborn+infant+using+phase+resolved+optical+spectroscopy.">Optical pathlength measurements on adult head, calf and forearm and the head of the newborn infant using phase resolved optical spectroscopy.</a> Physics in Medicine and Biology. 40 (2), 295-304 (1995).</li><li>Brigadoi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+artifacts+in+functional+near-infrared+spectroscopy:+A+comparison+of+motion+correction+techniques+applied+to+real+cognitive+data.">Motion artifacts in functional near-infrared spectroscopy: A comparison of motion correction techniques applied to real cognitive data.</a> NeuroImage. 85, 181-191 (2014).</li><li>Lopez-Calderon, J., Luck, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERPLAB:+an+open-source+toolbox+for+the+analysis+of+event-related+potentials.">ERPLAB: an open-source toolbox for the analysis of event-related potentials.</a> Frontiers in Human Neuroscience. 8, 213 (2014).</li></ol>

The authors have nothing to disclose.&#8195;

In this protocol, combined EEG and fNIRS recordings were performed to examine the brain activation patterns involving an event-related Flanker paradigm by recording the neural signals of the whole brain and concurrent hemodynamic responses of the prefrontal cortex. The ERP results showed that N200 at Fz was able to significantly distinguish the congruent and incongruent conditions (P=0.037). Meanwhile, the HbO signals in SFC (channels 21) also exhibited a significant difference between the congruent and incongruent conditions, which demonstrated the important role of the ability to suppress responses that involved the brain cognitive function associated with the Flanker task (PFDR = 0.041).
In addition, N200 at Fz showed a significant correlation with the hemodynamic response in the SFC (channel 21) for the incongruent condition although this was not the case for the congruent one. The brain activation in the prefrontal cortex is strongly correlated with high cognitive functions, which can be easily identified by fNIRS with the high signal-to-noise ratio in the spatial domain. However, the neural activity (N200) detected by EEG associated with the same Flanker task is mostly revealed in the parietal cortex with high sensitivity and high temporal resolution. N200 at Fz exhibited the cognitive difference between the two conditions, whereas fNIRS signals illustrated the difference of suppression function in the prefrontal region between the two conditions. It was discovered that the cognition showed a significant relationship with executive control during the Flanker task. This might be the main reason why the N200 at Fz exhibited a significant correlation with the hemodynamic response in SFC.
In this protocol, we described how to conduct fused EEG and fNIRS recordings and how to analyze the event-related potential and measure the hemoglobin concentration changes in the prefrontal cortex. The synchronization of different setups is an essential concern for the fusion of two hardware systems. Meanwhile, the event-related trigger is also the crucial mark for the task design of concurrent EEG and fNIRS recordings.
Combined EEG and fNIRS recordings are promising techniques for the investigation of the neural mechanisms underlying various cognitive tasks. In summary, we successfully acquired concurrent EEG and fNIRS data during a Flanker task. The findings indicated that the fNIRS hemodynamic response and ERP component N200 were significantly correlated, which exhibited different perspectives of the cognitive mechanism associated with the Flanker task. The multi-modal neuroimaging results support an essential role of combined EEG and fNIRS technique in contributing to brain cognition with different latencies and activation regions, which paves a new avenue for improving the understanding of the neural mechanisms of Flanker task.

This study aims to develop a working protocol to reveal the neural activation pattern underlying the Flanker task by using fused EEG and fNIRS neuroimaging techniques. Interestingly, the concurrent fNIRS-EEG recordings allow for the inspection of the relationship between the hemodynamic signals in the prefrontal cortex and various event-related potential (ERP) components of the whole brain associated with the Flanker task.
The integration of various noninvasive neuroimaging modalities including functional near-infrared spectroscopy (fNIRS), electroencephalography (EEG), and functional magnetic resonance imaging (fMRI) is essential to improve the understanding of where and when information processing is taking place in the brain1,2,3. Additionally, there is the potential to combine fNIRS and EEG to examine the relationship between local neural activity and subsequent changes in hemodynamic responses, in which EEG and fNIRS can be complementary in revealing the neural mechanism of human brain cognitive function. fNIRS is a vascular-based functional neuroimaging technique that relies on the hemodynamic responses to infer brain activation. fNIRS measures the relative oxyhemoglobin (HbO) and deoxyhemoglobin (HbR) concentration changes in the cerebral cortex, which plays an important role in the study of cognitive processing3,4,5,6,7. According to the neurovascular and neurometabolic coupling mechanism8, the change of local neural activity associated with cognitive processing is generally accompanied by subsequent alterations in the local blood flow and blood oxygen with a delay of 4-7 seconds. It is shown that the neurovascular coupling is likely a power transducer, which integrates the fast dynamics of neural activity into the vascular input of slow hemodynamics9. Specifically, fNIRS is mostly used for inspecting the neurovascular activity in the frontal lobe, especially the prefrontal cortex that is responsible for high cognitive functions, such as executive functions10,11,12, reasoning and planning13, decision making14, and social cognition and moral judgment15. However, the hemodynamic responses measured by fNIRS only indirectly capture the neural activity with a low temporal resolution, whereas EEG can offer temporally fine and direct measures of neural activities. Consequently, the combination of EEG and fNIRS recording can identify more features and reveal more information associated with the functioning of the brain.
More importantly, the multi-modal acquisition of EEG and fNIRS signals has been conducted to inspect the brain activation underlying various cognitive tasks16,17,18,19,20,21,22 or brain-computer interface23,24. In particular, concurrent ERP (event-related potential) and fNIRS recordings were carried out based on the event-related auditory oddball paradigm1, in which fNIRS can identify the hemodynamic changes in the frontotemporal cortex several seconds after the appearance of P300 component. Horovitz et al. also demonstrated the simultaneous measurements of fNIRS signals and the P300 component during a semantic processing task25. Interestingly, previous studies based on simultaneous EEG and fNIRS recordings showed that P300 during oddball stimuli exhibited a significant correlation with fNIRS signals26. It was discovered that the multi-modal measures have the potential to reveal the comprehensive cognitive neural mechanism based on the event-related paradigm26. Besides the oddball task, the Flanker task associated with ERP component N200 is also an important paradigm, which can be used for the investigation of cognitive ability detection and evaluation with healthy controls and patients with various disorders. Specifically, N200 was a negative component that peaks 200-350 ms from the anterior cingulated cortex frontal27 and superior temporal cortex28. Although previous studies examined the relationship between the superior frontal cortex and alpha oscillation in the Flanker task29, the correlation between the N200 amplitude and the hemodynamic responses during the Flanker task has not been explored.
In this protocol, a home-made EEG/fNIRS patch based on standard EEG cap was utilized for the concurrent EEG and fNIRS recordings. The arrangements of optodes/electrodes with support were achieved through the placement of fNIRS optodes fused into the EEG cap. The simultaneous EEG and fNIRS data acquisitions were carried out with the same stimuli tasks generated by E-prime software. We hypothesize that ERP components associated with the Flanker task can exhibit a significant correlation with the hemodynamic responses in the prefrontal cortex. Meanwhile, the combined ERP and fNIRS recordings can extract multiple signal indicators to identify the brain activation patterns with enhanced accuracy. To test the hypothesis, the fNIRS setup and EEG machine were integrated to reveal the complex neural cognition mechanism corresponding to the event-related Flanker task.

<table border="0" cellpadding="0" cellspacing="0" style="width:605px;" width="606">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EEG cap</td>
			<td style="width:104px;">EASYCAP GmbH</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EEG system</td>
			<td style="width:104px;">BioSemi</td>
			<td style="width:119px;">-</td>
			<td>-</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fNIRS system</td>
			<td style="width:104px;">TechEn</td>
			<td style="width:119px;">-</td>
			<td>CW6 System</td>
		</tr>
	</tbody>
</table>

conducting concurrent electroencephalography and functional near-infrared spectroscopy recordings with a flanker task

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by inspecting the relationship between the neural and hemodynamic signals. EEG is an electrophysiological technology that can measure the rapid neuronal activity of the cortex, whereas fNIRS relies on the hemodynamic responses to infer brain activation. The combination of EEG and fNIRS neuroimaging techniques can identify more features and reveal more information associated with the functioning of the brain. In this protocol, fused EEG-fNIRS measurements were performed for concurrent recordings of evoked-electrical potentials and hemodynamic responses during a Flanker task. In addition, the critical steps for setting up the hardware and software system as well as the procedures for data acquisition and analysis were provided and discussed in detail. It is expected that the present protocol can pave a new avenue for improving the understanding of the neural mechanisms underlying various cognitive processes by using the EEG and fNIRS signals.

Figure 2 shows the HbO signals for all channels while Figure 3 displays the ERPs at Fz and FCz for the two conditions of the Flanker task. Figure 4 illustrated the Pearson correlation analysis results showed that the fNIRS signals in SFC exhibited a significant correlation with the ERP N200 component at Fz for the incongruent condition (P&#60;0.05). However, this is not the case for the congruent conditions (P&#62;0.05).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60669/60669fig1.jpg" /> 
Figure 1. fNIRS headset placement and channel configuration. The digitized optodes layout are converted into the MNI coordinate system and then overlapped along the brain cortex <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60669/60669fig1large.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/60669/60669fig2.jpg" /> 
Figure 2. HbO signals for all channels associated with the Flanker task. The pink curves denote the incongruent condition while the green ones indicate the congruent condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60669/60669fig2large.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/60669/60669fig3.jpg" /> 
Figure 3. ERP signals for Fz and FCz electrodes. The black curves define the incongruent condition while the red ones denote the congruent condition. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60669/60669fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60669/60669fig4.jpg" /> 
Figure 4: Correlation between the ERP N200 and HbO signals along the superior frontal cortex (SFC) for the incongruent condition. The regression coefficient between the two measurements is 0.59, p = 0.027.&#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60669/60669fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task at JoVE.com

Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task

The present protocol describes how to perform concurrent EEG and fNIRS recordings and how to inspect the relationship between the EEG and fNIRS data.&#160;

Concurrent EEG and fNIRS recordings offer an excellent opportunity to gain a full understanding of the neural mechanism of cognitive processing by ...

conducting-concurrent-electroencephalography-functional-near-infrared

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.6K Views.  University of Macau. The present protocol describes how to perform concurrent EEG and fNIRS recordings and how to inspect the relationship between the EEG and fNIRS data. 

Video: Conducting Concurrent Electroencephalography and Functional Near-Infrared Spectroscopy Recordings with a Flanker Task

Functional Mapping with Simultaneous MEG and EEG

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the processing of simple sensory stimuli. We will use somatosensory stimuli to locate the hand somatosensory areas, auditory stimuli to locate the auditory cortices, visual stimuli in four quadrants of the visual field to locate the early visual areas. These type of experiments are used for functional mapping in epileptic and brain tumor patients to locate eloquent cortices. In basic neuroscience similar experimental protocols are used to study the orchestration of cortical activity. The acquisition protocol includes quality assurance procedures, subject preparation for the combined MEG/EEG study, and acquisition of evoked-response data with somatosensory, auditory, and visual stimuli. We also demonstrate analysis of the data using the equivalent current dipole model and cortically-constrained minimum-norm estimates. Anatomical MRI data are employed in the analysis for visualization and for deriving boundaries of tissue boundaries for forward modeling and cortical location and orientation constraints for the minimum-norm estimates.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to map brain areas involved in the processing of simple sensory stimuli.

We use magnetoencephalography (MEG) and electroencephalography (EEG) to locate and determine the temporal evolution in brain areas involved in the ...

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 ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy (NIRS) Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in remote locations. This paper shows the development of field functional Near Infrared Spectroscopy (fNIRS) imaging to the study of child language, reading, and cognitive development in a rural village setting of C&#244;te d&#39;Ivoire. Innovation in methods and the development of culturally appropriate neuroimaging protocols allow&#160;a first-time look into the brain&#39;s development and children&#39;s learning outcomes in understudied environments. This paper demonstrates protocols for transporting and setting up a mobile laboratory, discusses considerations for field versus laboratory neuroimaging, and presents a guide for developing neuroimaging consent procedures and building meaningful long-term collaborations with local government and science partners. Portable neuroimaging methods can be used to study complex child development contexts, including the impact of significant poverty and adversity on brain development. The protocol presented here has been developed for use in C&#244;te d&#39;Ivoire, the world&#39;s primary source of cocoa, and where reports of child labor in the cocoa sector are common. Yet, little is known about the impact of child labor on brain development and learning. Field neuroimaging methods have the potential to yield new insights into such urgent issues, and the development of children globally.

Neuroimaging Field Methods Using Functional Near Infrared Spectroscopy NIRS Neuroimaging to Study Global Child Development: Rural Sub-Saharan Africa

Portable neuroimaging approaches (functional Near Infrared Spectroscopy) provide advances to the study of the brain in previously inaccessible regions; here, rural C&#244;te d&#39;Ivoire. Innovation in methods and development of culturally-appropriate neuroimaging protocols permits novel study of the brain&#39;s development and children&#39;s learning outcomes in environments with significant poverty and adversity.

Portable neuroimaging approaches provide new advances to the study of brain function and brain development with previously inaccessible populations and in ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design

Neuroimaging studies play a pivotal role in the evaluation of pre- vs. post-interventional neurological conditions such as in rehabilitation and surgical treatment. Among the many neuroimaging technologies used to measure brain activity, functional near-infrared spectroscopy (fNIRS) enables the evaluation of dynamic cortical activities by measuring the local hemoglobin levels similar to functional magnetic resonance imaging (fMRI). Also, due to lesser physical restriction in fNIRS, multiple variants of sensorimotor tasks can be evaluated. Many laboratories have developed several methods for fNIRS data analysis; however, despite the fact that the general principles are the same, there is no universally standardized method. Here, we present the qualitative and comparative analytic methods of data obtained from a multi-channel fNIRS experiment using a block design. For qualitative analysis, we used a software for NIRS as a mass-univariate approach based on the generalized linear model. The NIRS-SPM analysis shows qualitative results for each session by visualizing the activated area during the task. In addition, the non-invasive three-dimensional digitizer can be used to estimate the fNIRS channel locations relative to the brain. To corroborate the NIRS-SPM findings, the amplitude of the changes in hemoglobin levels induced by the sensorimotor task can be statistically analyzed by comparing the data obtained from two different sessions (before and after intervention) of the same study subject using a multi-channel hierarchical mixed model. Our methods can be used to measure the pre- vs. post-intervention analysis in a variety of neurological disorders such as movement disorders, cerebrovascular diseases, and neuropsychiatric disorders.

We describe the analysis of continuous-wave functional near-infrared spectroscopy experiment using a block design with a sensorimotor task. To increase the reliability of the data analysis, we used the qualitative general linear model-based statistical parametric mapping and the comparative hierarchical mixed models for multi-channels.

Neuroimaging studies play a pivotal role in the evaluation of pre- vs. post-interventional neurological conditions such as in rehabilitation and surgical ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...