This work was partly supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) 18K08956 and a fund from the Central Research Institute of Fukuoka University (No. 201045).

This study was approved by the institutional review board (IRB) of the Fukuoka University, Japan (IRB No. 2017M017). Prior to participation, all patients provided written informed consent.
1. Preparation of the fNIRS experiment
NOTE: A multi-channel continuous-wave laser-based NIRS system for this experiment was used. The wavelengths of the near-infrared light were 780 nm, 805 nm, and 830 nm, and the sampling rate was set at 7.8 Hz. The time and spatial resolution (distances between the light emitter and detector probe) were 0.13 s and 3.0 cm, respectively.
<ol>
	<li>Set the fNIRS device in a dark noiseless place. Conduct experiments at room temperature. Start the fNIRS instrument 30 min before the experiment. 
	NOTE: fNIRS systems are used under controlled temperature and humidity conditions to ensure stable operation of the equipment26.</li>
	<li>Use a whole head caps for fNIRS recording and attach the head cap on the subject&#39;s head such that the position corresponding to the central (Cz) of international 10-20 system is located at the holder No. 245 of the head cap. (Figure 1).</li>
	<li>Attach the marking sticker to reference locational points: the nasion (Nz), right external auditory meatus (AR), and left external auditory meatus (AL). 
	NOTE: Because the three-dimensional (3D) coordinates are read around the positions of the Nz, AR, AL marking sticker and Cz holder, it is necessary to attach the marking sticker before taking a picture.</li>
	<li>After the calibration of a high-resolution digital camera for spatial registration, take pictures of the subject&#39;s head with the probe location while showing the reference points (Cz, Nz, AR, and LR) from 15 perspectives. 
	NOTE: Please take a picture before placing the probe. If a picture is taken after placing the probe, the marker landmark may be hidden by the probe and wiring cord. As recommended by the manufacturer, after taking 12 pictures 30&#176; diagonally forward to the right of the subject, take three or more pictures slightly above so that the Cz (holder No. 245) appears in the picture. This is because it is easy to make it three-dimensional when capturing a total of 15 or more shots.</li>
	<li>Carefully separate the subject&#39;s hair that interferes with the optode using a light-emitting diode (LED)-lit plastic rod to attach the probe. Arrange the probe so that the optodes are attached at a minimal distance from the scalp surface and in contact with the scalp. 
	NOTE: Check carefully whether there is any pressure or discomfort for the patient due to the attachment of the optodes, because of increasing strength of the systemic confounders associated with autonomic nervous system activation23.</li>
	<li>Arrange the 48-channel system with 32 optodes (16 light sources and 16 detectors; 4 x 4 array for each hemisphere) to a head cap bilaterally over the frontal and parietal areas as regions of interest (Figure 2).</li>
	<li>Start and use the 3D-digitizer software to determine the spatial registration.</li>
	<li>After scanning the picture data of an entire head, determine the spatial coordinate of each patient by auto-measuring and save as the Origin and the Others file (*CSV file). 
	&#8203;NOTE: If the coordinate points could not be detected from the images using the automatic measurement, input the adjustment manually.</li>
</ol>
2. Run the experiment
<ol>
	<li>Select a block design for the experiment, and the task can be any movement of interest for the study such as hand opening/closing, finger tapping, etc. In our previous study, the task was the robot-assisted elbow movements15. 
	NOTE: Each cycle is made of three blocks (15 s of rest - 15 s of task - 15 s of rest), and each patient completes seven cycles in each session.</li>
	<li>Make the participant wait in a comfortable position until the starting signal. Instruct the subject to close their eyes during the rest and the task.</li>
	<li>Give start and stop cues (i.e., &#34;Repeat flexion and extension of the elbow&#34;, &#34;Stop and relax&#34;). 
	NOTE: Do not speak during measurements. Carefully check for artifacts on the monitor screen during measurements.</li>
	<li>Perform the block design task in the same posture. Upright posture with standing or sitting position is desirable not to distort the headset. 
	NOTE: If the patient feels uncomfortable after wearing the head holder for an extended period of time, remove or loosen the probe during robot-assisted exercise.</li>
	<li>After completing the NIRS measurement, remove the head holder and marking sticker to end the experiment. 
	&#8203;NOTE: Carefully check for skin damage to the scalp due to prolonged wearing.</li>
</ol>
3. Qualitative GLM analysis using NIRS-SPM software
<ol>
	<li>Start the NIRS-SPM on the MATLAB software. Convert the data file related to the change in HbO2 and HHb concentration acquired from the NIRS device to the file format for NIRS-SPM analysis.</li>
	<li>Choose the using NIRS system option from the pop-up menu. Select the load button and choose the convert HbO2 and HHb concentration change options.</li>
	<li>Detect the spatial registration of NIRS channel location. Select the Stand-alone checkbox and then select the With 3D Digitizer checkbox.</li>
	<li>Within From Real Coordinates to MNI Space, use the dialog box to choose _origin.CSV referring to the coordinate reference point file, and _others. CSV referring to the coordinate probes/channels file.</li>
	<li>Select the Registration button. Choose the points to proceed to spatial estimation, and click on the OK button. Click on the Project MNI coordinate to Rendered Brain button. 
	NOTE: The spatial position of the NIRS channel locations is estimated on the basis of Montreal Neurological Institute (MNI) brain template.</li>
	<li>Select the Dorsal View option, and click on the Save button.</li>
	<li>In the Specify the 1st Level section, select the NIRS data filename and SPM directory. Select the hemoglobin checkbox; HbO2 or HHb. Highlight the Specify design&#39; option and select the Sec option. Highlight the Number of Condition/Trials options and enter the number 7.</li>
	<li>Highlight the Vector of Onset and Duration[s] options and enter a vector of onset multiplied by the duration of the experimental conditions as follows. 
	NOTE: In this case, the vector of onset times should be specified as [15:45:285] or [15 60 105 150 195 240 285]. The vector of duration should be specified as [15* ones(7,1)] or [15 15 15 15 15 15 15].</li>
	<li>For detrending, select the Wavelet-MDL button. Use the precoloring method: low-pass filter and select the hrf button, and correct for serial correlation, then select the none button.</li>
	<li>In estimating the temporal correlations, check the Individual Analysis. 
	NOTE: Save the spatial localization of fNIRS channel positions in the individual MNI coordinate systems as a text file. Similarly, save the map based on the individual Brodmann area (BA) as a text file.</li>
	<li>In estimating the temporal correlations, check the Group Analysis. The NIRS-SPM aligned the mean optode positions of the number of participants according to the MNI standardized brain coordinate system.</li>
	<li>Compute the activation map based on the changes in hemoglobin level for the standardized brain. HbO2 and HHb levels were considered significant at an uncorrected threshold of p &#60; 0.01. 
	&#8203;NOTE: Left/right information was flipped in the right-affected sides for group analysis.</li>
</ol>
4. Multi-channel comparative analysis based on hierarchical mixed model
<ol>
	<li>Start the SAS software. Convert the text document (.TXT) of concentration changes of HbO2 and HHb in the NIRS data file processed with a low pass filter (cut-off frequency was set at 0.1 Hz) to the spreadsheet software comma separated values file (.CSV).</li>
	<li>Create the Import SAS data (.sas7bdat) using the program.</li>
	<li>Output the Import file with the following command, libname out &#34;Import file&#34;</li>
	<li>Output the pre- vs. post-intervention file for each subject, run the following commands in the Analysis SAS. (Figure 3) During the creation of the import file, give a name such that can identify the subject information and pre- vs. post-intervention (e.g., id1 pre, id1 post&#8230;)</li>
	<li>Run the pre- and post-intervention data command for each channel (ch1-48; HbO2 and HHb) as follows (Figure 4).</li>
	<li>Based on the data obtained from the output results, input the pre- vs. post-intervention differences in change (difference at on-task and rest), rest, and on-task values (estimated values, upper limits, and lower limits) of each channel in the spreadsheet workbook file (.xlsx).</li>
	<li>Similarly, input the numerator and denominator degrees of freedom, F value, and P value of the interact item of the fixed-effect Type 3 test to the spreadsheet workbook file (.xlsx).</li>
	<li>To control the false discovery rate (FDR) in multi-channel testing, use the Benjamin and Hochberg methods27 and control the FDR at a p-value &#60; 0.01.</li>
</ol>

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The authors have no conflicts of interest relevant to this study to disclose.

In our group analytic methods for fNIRS, in addition to performing an imaging analytic method by qualitative t-statistic mappings, we compared pre- vs. post-intervention (robot-assisted exercise) using the comparative multichannel analysis. For qualitative analysis, we used the NIRS-SPM software as a mass-univariate approach based on the generalized linear model. The NIRS-SPM analysis shows qualitative results of each session by visualizing the activated area during the task. Moreover, the information of the non...

Neurorehabilitation plays an important role in the functional recovery following sensorimotor disturbance. To clarify the mechanisms of neuroplasticity-associated functional recovery, various neuroimaging technologies have been used, such as functional magnetic resonance imaging (fMRI), positron emission tomography (PET), electroencephalography (EEG), and functional near-infrared spectroscopy (fNIRS). Different imaging modalities have different advantages and disadvantages. Although the fMRI is the most typical device, it is affected by magnetic fields, has a high cost, high physical restriction, and limited sensorimotor tasks1,2,3,4. The fNIRS device stands out as a noninvasive optical neuroimaging and has a relatively lower spatial resolution, but it has a better temporal resolution than fMRI4. fNIRS is suitable when verifying treatment effects because it compares the pre- versus post-intervention effects, has dynamic motor tasks, is portable, and functions more in natural environments than fMRI1,2,4. NIRS has been reported to be more suitable in the fields of cerebrovascular disease, epileptic disorders, severe brain injury, Parkinson&#39;s disease, and cognitive impairment1,5. With regard to sensorimotor tasks, it is widely used in gait and standing balance6,7,8, upper limb function (hand grasping, finger tapping)8,9, complex motor skill training10,11, robotics12,13,14,15, and brain-computer interface16,17,18. The fNIRS is based on the principles of optical neuroimaging and neurovascular coupling, which measure cortical metabolic activity, increased blood flow, and consequently cortical activity as secondary signals19. fNIRS signals have been reported to have strong correlations with signals of blood oxygen level-dependent fMRI20. A continuous-wave fNIRS uses the modified Beer-Lambert law&#160;to determine the changes in oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (HHb) cortical concentration levels based on measured changes in broadband near-infrared light attenuation21,22. Because it was not possible to measure the differential path-length factor (DPF) using the continuous-wave NIRS system, we assumed that the DPF was constant and that hemoglobin signal changes were denoted in arbitrary units of millimole-millimeter (mM x mm)2,18.
The fNIRS experiments need to select the most adequate methods including the probe settings, the experiment designs, and the analysis methods. Regarding the probe setting, the international 10-20 method used in EEG measurement is the setting standard used by many researchers in neuroimaging. In recent years, coordinate settings based on the standard brain on the basis of Montreal Neurological Institute (MNI) coordinates have been used. The experiment uses a block design, generally used for sensorimotor tasks, and an event-related design. This is a method of comparing changes in hemoglobin concentration at rest and during tasks; HbO2 concentration levels increase and HHb concentration levels decrease with changes in cerebral blood flow associated with task-dependent cortical activity. Although there are various analysis methods, the NIRS-SPM free software enables an analysis similar to the statistical parametric mapping (SPM) of fMRI. The treatment of NIRS data uses a mass-univariate approach based on the general linear model (GLM). When performing task-dependent brain activity analysis, the fNIRS measurements can be affected by evoked or non-evoked neuronal activity and systemic physiological interferences (heart rate, blood pressure, breathing rate, and autonomic nervous system activity) in the cerebral and extracerebral compartment23. Therefore, pre-analysis processing, filtering, wavelet conversion, and principal component analysis are useful23. Regarding filtering and artifacts of the data processing using the NIRS-SPM, low-pass filtering9 and the wavelet minimum description length (Wavelet-MDL)24 detrending were used to overcome the motion or other sources of noise/artifact. For details of this analytic method, refer to the report of Ye et al.25. Although there are reports using only SPM, it is only a qualitative index by image analysis, and due to the low spatial resolution of NIRS, extreme caution is required for group analysis. Moreover, when the DPF is constant, numerical comparisons between channels and individuals should not be performed, but the difference in the changes in each channel can be verified. Based on the above conditions, in order to supplement the NIRS-SPM group analysis results, we used the original analysis method for multi-channel analysis after improving the accuracy of spatial registration. This multi-channel analysis compared the amplitude of the change in HbO2 and HHb levels between the rest and on-task periods at each channel before and immediately after treatment using hierarchical mixed models with fixed interventions (before or after), fixed periods (rest or on-task), and random individual effects.
In this way, there are several fNIRS measurement and analysis methods; however, no standard method has been established. In this paper, we introduce our methods, qualitative GLM-based statistical parametric mapping and the comparative multi-level hierarchical mixed model, to analyze data obtained from a multi-channel fNIRS experiment of pre- vs. post-intervention using a block design with sensorimotor tasks.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D-digitizer software</td>
			<td>TOPCON</td>
			<td>-</td>
			<td>NS-1000 software ver.1.50</td>
		</tr>
		<tr>
			<td>NIRS system</td>
			<td>Shimadzu</td>
			<td>-</td>
			<td>FOIRE-3000</td>
		</tr>
		<tr>
			<td>Robot</td>
			<td>CYBERDYNE</td>
			<td>-</td>
			<td>Single-joint type Hybrid Assitive Limb (HAL-SJ)</td>
		</tr>
	</tbody>
</table>

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.

Herein, we introduce the robot-assisted rehabilitation that our group is currently working on: the biofeedback effects on upper limb motor deficit in patients with acute stroke. We included 10 consenting stroke patients (mean age: 66.8 &#177; 12.0 years; two women and eight men) who were admitted to our hospital. At the subacute stroke stage, more than 2 weeks after the onset, we evaluated the motor-related cortical activity of these patients using an fNIRS system before and immediately a...

Watch this Scientific Journal Video about Qualitative and Comparative Cortical Activity Data Analyses from a Functional Near-Infrared Spectroscopy Experiment Applying Block Design at JoVE.com

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

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 ...

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3.6K Views.  Fukuoka University. 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.

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

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 Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Recording Human Electrocorticographic (ECoG) Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), magnetoencephalography or functional magnetic-resonance imaging. These techniques have either inherently low temporal or low spatial resolution, and suffer from low signal-to-noise ratio and/or poor high-frequency sensitivity. Thus, they are suboptimal for exploring the short-lived spatio-temporal dynamics of many of the underlying brain processes. In contrast, the invasive technique of electrocorticography (ECoG) provides brain signals that have an exceptionally high signal-to-noise ratio, less susceptibility to artifacts than EEG, and a high spatial and temporal resolution (i.e., &lt;1 cm/&lt;1 millisecond, respectively). ECoG involves measurement of electrical brain signals using electrodes that are implanted subdurally on the surface of the brain. Recent studies have shown that ECoG amplitudes in certain frequency bands carry substantial information about task-related activity, such as motor execution and planning1, auditory processing2 and visual-spatial attention3. Most of this information is captured in the high gamma range (around 70-110 Hz). Thus, gamma activity has been proposed as a robust and general indicator of local cortical function1-5. ECoG can also reveal functional connectivity and resolve finer task-related spatial-temporal dynamics, thereby advancing our understanding of large-scale cortical processes. It has especially proven useful for advancing brain-computer interfacing (BCI) technology for decoding a user's intentions to enhance or improve communication6 and control7. Nevertheless, human ECoG data are often hard to obtain because of the risks and limitations of the invasive procedures involved, and the need to record within the constraints of clinical settings. Still, clinical monitoring to localize epileptic foci offers a unique and valuable opportunity to collect human ECoG data. We describe our methods for collecting recording ECoG, and demonstrate how to use these signals for important real-time applications such as clinical mapping and brain-computer interfacing. Our example uses the BCI2000 software platform8,9 and the SIGFRIED10 method, an application for real-time mapping of brain functions. This procedure yields information that clinicians can subsequently use to guide the complex and laborious process of functional mapping by electrical stimulation.
Prerequisites and Planning:
Patients with drug-resistant partial epilepsy may be candidates for resective surgery of an epileptic focus to minimize the frequency of seizures. Prior to resection, the patients undergo monitoring using subdural electrodes for two purposes: first, to localize the epileptic focus, and second, to identify nearby critical brain areas (i.e., eloquent cortex) where resection could result in long-term functional deficits. To implant electrodes, a craniotomy is performed to open the skull. Then, electrode grids and/or strips are placed on the cortex, usually beneath the dura. A typical grid has a set of 8 x 8 platinum-iridium electrodes of 4 mm diameter (2.3 mm exposed surface) embedded in silicon with an inter-electrode distance of 1cm. A strip typically contains 4 or 6 such electrodes in a single line. The locations for these grids/strips are planned by a team of neurologists and neurosurgeons, and are based on previous EEG monitoring, on a structural MRI of the patient's brain, and on relevant factors of the patient's history. Continuous recording over a period of 5-12 days serves to localize epileptic foci, and electrical stimulation via the implanted electrodes allows clinicians to map eloquent cortex. At the end of the monitoring period, explantation of the electrodes and therapeutic resection are performed together in one procedure.
In addition to its primary clinical purpose, invasive monitoring also provides a unique opportunity to acquire human ECoG data for neuroscientific research. The decision to include a prospective patient in the research is based on the planned location of their electrodes, on the patient's performance scores on neuropsychological assessments, and on their informed consent, which is predicated on their understanding that participation in research is optional and is not related to their treatment. As with all research involving human subjects, the research protocol must be approved by the hospital's institutional review board. The decision to perform individual experimental tasks is made day-by-day, and is contingent on the patient's endurance and willingness to participate. Some or all of the experiments may be prevented by problems with the clinical state of the patient, such as post-operative facial swelling, temporary aphasia, frequent seizures, post-ictal fatigue and confusion, and more general pain or discomfort.
At the Epilepsy Monitoring Unit at Albany Medical Center in Albany, New York, clinical monitoring is implemented around the clock using a 192-channel Nihon-Kohden Neurofax monitoring system. Research recordings are made in collaboration with the Wadsworth Center of the New York State Department of Health in Albany. Signals from the ECoG electrodes are fed simultaneously to the research and the clinical systems via splitter connectors. To ensure that the clinical and research systems do not interfere with each other, the two systems typically use separate grounds. In fact, an epidural strip of electrodes is sometimes implanted to provide a ground for the clinical system. Whether research or clinical recording system, the grounding electrode is chosen to be distant from the predicted epileptic focus and from cortical areas of interest for the research. Our research system consists of eight synchronized 16-channel g.USBamp amplifier/digitizer units (g.tec, Graz, Austria). These were chosen because they are safety-rated and FDA-approved for invasive recordings, they have a very low noise-floor in the high-frequency range in which the signals of interest are found, and they come with an SDK that allows them to be integrated with custom-written research software. In order to capture the high-gamma signal accurately, we acquire signals at 1200Hz sampling rate-considerably higher than that of the typical EEG experiment or that of many clinical monitoring systems. A built-in low-pass filter automatically prevents aliasing of signals higher than the digitizer can capture. The patient's eye gaze is tracked using a monitor with a built-in Tobii T-60 eye-tracking system (Tobii Tech., Stockholm, Sweden). Additional accessories such as joystick, bluetooth Wiimote (Nintendo Co.), data-glove (5th Dimension Technologies), keyboard, microphone, headphones, or video camera are connected depending on the requirements of the particular experiment.
Data collection, stimulus presentation, synchronization with the different input/output accessories, and real-time analysis and visualization are accomplished using our BCI2000 software8,9. BCI2000 is a freely available general-purpose software system for real-time biosignal data acquisition, processing and feedback. It includes an array of pre-built modules that can be flexibly configured for many different purposes, and that can be extended by researchers' own code in C++, MATLAB or Python. BCI2000 consists of four modules that communicate with each other via a network-capable protocol: a Source module that handles the acquisition of brain signals from one of 19 different hardware systems from different manufacturers; a Signal Processing module that extracts relevant ECoG features and translates them into output signals; an Application module that delivers stimuli and feedback to the subject; and the Operator module that provides a graphical interface to the investigator.
A number of different experiments may be conducted with any given patient. The priority of experiments will be determined by the location of the particular patient's electrodes. However, we usually begin our experimentation using the SIGFRIED (SIGnal modeling For Realtime Identification and Event Detection) mapping method, which detects and displays significant task-related activity in real time. The resulting functional map allows us to further tailor subsequent experimental protocols and may also prove as a useful starting point for traditional mapping by electrocortical stimulation (ECS).
Although ECS mapping remains the gold standard for predicting the clinical outcome of resection, the process of ECS mapping is time consuming and also has other problems, such as after-discharges or seizures. Thus, a passive functional mapping technique may prove valuable in providing an initial estimate of the locus of eloquent cortex, which may then be confirmed and refined by ECS. The results from our passive SIGFRIED mapping technique have been shown to exhibit substantial concurrence with the results derived using ECS mapping10.
The protocol described in this paper establishes a general methodology for gathering human ECoG data, before proceeding to illustrate how experiments can be initiated using the BCI2000 software platform. Finally, as a specific example, we describe how to perform passive functional mapping using the BCI2000-based SIGFRIED system.

Recording Human Electrocorticographic ECoG Signals for Neuroscientific Research and Real-time Functional Cortical Mapping

We present a method for collecting electrocorticographic signals for research purposes from humans who are undergoing invasive epilepsy monitoring. We show how to use the BCI2000 software platform for data collection, signal processing and stimulus presentation. Specifically, we demonstrate SIGFRIED, a BCI2000-based tool for real-time functional brain mapping.

Neuroimaging studies of human cognitive, sensory, and motor processes are usually based on noninvasive techniques such as electroencephalography (EEG), ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. 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 ...

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.

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 ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

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, ...