This work was supported by Incheon National University (International Cooperative) for Sunggu Yang.

All animal-handling procedures were approved by the Institutional Animal Care and Use Committee of the Incheon National University (INU-ANIM-2017-08).
1. Animal preparation for surgery
NOTE: Use Sprague Dawley Rat (8-10 weeks old) without the sex bias for this experiment.
<ol>
	<li>Anesthetize the rat with 90 mg/kg ketamine and 10 mg/kg xylazine cocktail intraperitoneally. To maintain the desired depth of anesthesia throughout the surgery, provide a supplemental 45 mg/kg ketamine and 5 mg/kg xylazine cocktail when the rat shows signs of waking up.</li>
	<li>Confirm that the rat is under deep anesthesia and regularly check body reflections such as toe pinch, tail pinch, and corneal reflex.</li>
	<li>Shave the fur between the eyes and the back of the ears using a trimmer.</li>
	<li>Apply an ophthalmic ointment onto the eyes to prevent them from drying out.</li>
</ol>
2. Surgery for cortical surface exposure
<ol>
	<li>Fix the rat head on the stereotaxic apparatus with a stereotaxic adaptor. To maintain the body temperature of 37 &#176;C during the surgery, place the rat on a temperature-controlled heating pad.</li>
	<li>Sterilize the shaved area with alternating scrubs of alcohol and povidone-iodine three times.</li>
	<li>Use forceps to grasp the scalp firmly and inject 0.1 mL of lidocaine (2%) with a syringe directly into the scalp to induce local anesthesia in the surgery area.</li>
	<li>Make a 2-3 cm long midline incision with a scalpel and pull apart the scalp to expose the skull.</li>
	<li>Clamp the scalp with mosquito forceps to expose the skull.</li>
	<li>Scratch the surface of the skull with forceps to remove the periosteum.</li>
	<li>Blunt dissect the muscles over the occipital skull to expose the cisterna magna above the axis on the top of the spinal cord.</li>
	<li>Incise the cisterna magna with the blade to drain the cerebrospinal fluid and put a sterile gauze inside the incision of the cisterna magna to absorb the cerebrospinal fluid constantly to prevent brain edema and minimize inflammation.</li>
	<li>Using a pencil, mark on the skull a rectangular window measuring 3 mm in the anteroposterior axis and 6 mm in the right lateral direction from the bregma of the right hemisphere. 
	NOTE: Marking must secure a 1 mm distance from the midline to avoid superior sagittal sinus rupture.</li>
	<li>Drill the marked area according to the stereotaxic coordinate and remove the skull with a bone rongeur.</li>
	<li>To remove the dura mater, bend the tip of the 26 G needle to 90&#176;, create a hole in the dura mater, lift the dura mater, insert forceps into that hole, and tear it with forceps.</li>
	<li>Place saline-wetted gauze on the somatosensory cortex to prevent it from drying out.</li>
</ol>
3. Preparation of graphene electrode array connected to the recording system
<ol>
	<li>Prepare a graphene electrode array with an omnetics connector.
	<ol>
		<li>Detach the graphene multielectrode array without causing damage by applying the saline solution.</li>
		<li>Remove the outer covering of the reference and ground wires from the connector.</li>
	</ol>
	</li>
	<li>Connect the head stage with the graphene electrode array to the connector.</li>
	<li>Plug the interface cable linked to the head stage into the recording system.</li>
	<li>Secure the graphene electrode array complex into the stereotaxic arm.</li>
	<li>To capture neural signals from all channels, position the array on the somatosensory cortex without any bending, following the predetermined stereotaxic coordinates.</li>
	<li>Place a reference wire underneath the tissue behind the occipital bone and connect the ground wire to the grounded optical table.</li>
</ol>
4. Physical stimulation and recording SEPs for mapping
<ol>
	<li>Open the neural signal recording software.</li>
	<li>Set the recording software environment: (1) set the sampling rate for SEPs and notch filter (60 or 50 Hz, a frequency of household electrical power) to remove the noise from the power line.</li>
	<li>For whisker mapping, bend the whisker with a fine stick.</li>
	<li>Constantly poke the forepaw, forelimb, hind paw, hindlimb, and trunk with a wooden stick for body mapping.</li>
	<li>Record neural signals in the data acquisition system for the indicated time.</li>
</ol>
5. Animal euthanasia
<ol>
	<li>After all the recording procedures, sacrifice the rats with anesthesia using &#62;5% isoflurane and perform cervical dissection.</li>
</ol>
6. SEP measurement for cortical mapping
<ol>
	<li>Open MATLAB code-named read_Intan_RHS2000_file.m for signal analysis. 
	NOTE: read_Intan_RHS2000_file.m can be downloaded from &#34;https://intantech.com/downloads.html?tabSelect=Software&#8221;.</li>
	<li>Click the Run button, select the recording file with the &#34;.rhs&#34; filename extension, and wait for the file to be processed and read.</li>
	<li>Enter the command &#34;plot (t, amplifier_data(&#34;channel number&#34;,:))&#34; to create a 2D line plot of the recording data, find the SEPs, and calculate the amplitude of SEPs in all channels. 
	NOTE: Enter the channel number at &#34;channel number.&#34; For example, &#34;plot(t, amplifier_data(1,:))&#34; creates channel 1&#39;s 2D line plot. In addition, when the experimenter calculates the amplitude of the response, choose the response recorded from each channel.</li>
	<li>Obtain data by coloring the grid with a different hue according to the amplitude of the SEPs. 
	NOTE: MATLAB command &#34;imagesc&#34; helps obtain a topographic map more quickly.</li>
</ol>

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We have nothing to disclose.

The presented protocol provides an in-depth, step-by-step process that explains how to access and map the somatosensory responses of rats using a graphene electrode array. The protocol-acquired data are SEPs that provide somatosensory information that is synaptically linked to each body part.
Several aspects of this protocol should be considered. When extracting cerebrospinal fluid to prevent brain edema and mitigate inflammation, it is crucial for the experimenter not to damage the brainstem located in front of the cisterna magna.
Facial whiskers provide tactile sensory information about the surroundings, such as a dark and narrow environment. Accordingly, rodent whiskers are well-developed enough to sense an object through the deflection directions, stimulus intensity, and location of the stimulated whiskers. The somatosensory cortex responds to the bending direction, intensity, and location of each whisker differently18,19. Therefore, all whiskers are stimulated with constant intensity and direction in this protocol.
This protocol cannot record signals evoked in deep brain structures as our graphene electrode array is mounted on the cortical surface. Thus, the experimenter cannot identify how the columnar network is hierarchically organized concerning neural responses.
This protocol is superior to previous recording methods because the graphene electrode array is less invasive, adaptable, and biocompatible12,13,14,15,16. Furthermore, the graphene electrode array has &#62;30 channels for recording signals, thus enabling faster cortical mapping than a single or tetrode electrode. This protocol can be further applied to other cortical areas whenever required15,20. The experimenter can place the electrode array on the auditory or visual cortex to extract auditory and visual information as the auditory or visual maps. Finally, this method can be implemented for chronic implantation and diagnosis of neural diseases, such as stroke, epilepsy, tinnitus, and Parkinson&#39;s disease.

A cortical map is a set of local patches representing response properties to sensorimotor stimuli in the cerebral cortex. They are a spatial formation of neural networks and enable prediction for perception and cognition. Therefore, cortical maps are useful in evaluating neural responses to external stimuli and processing sensorimotor information1,2,3,4. Invasive and noninvasive methods are available for cortical mapping. One of the most common invasive methods involves the use of intracortical (or penetrating) electrodes for mapping5,6,7,8.
Assessing the on-demand high-resolution cortical maps using penetrating electrodes has faced several obstacles. The method is too laborious to obtain a decent map and too invasive to implement for clinical use, prohibiting further development. More recent technologies such as electroencephalography (EEG), positron emission tomography (PET), magnetoencephalography (MEG), and functional magnetic resonance imaging (fMRI) have gained popularity because these are less invasive and reproducible. However, given their prohibitive costs and poor resolution, they are used in a limited number of cases9,10,11. Recently, flexible surface electrodes with superior signal reliability have attracted considerable attention. Graphene-based surface electrodes demonstrate long-term biocompatibility and mechanical flexibility, providing stable recordings in a convoluted brain12,13,14,15,16. Our group has recently developed a graphene-based multichannel array for high-resolution recording and site-specific neurostimulation on the cortical surface. This technology allows us to keep track of the cortical representations of sensory information for an extended period.
This article describes the steps involved in acquiring a brain map of the somatosensory cortex using a 30-channel graphene multielectrode array. To measure brain activity, a graphene electrode array is placed on the subdural area of the cortex, while the forepaw, forelimb, hind paw, hindlimb, trunk, and whiskers are stimulated with a wooden stick. The somatosensory-evoked-potentials (SEPs) are recorded for somatosensory areas. This protocol can also be applied to other brain areas, such as the auditory, visual, and motor cortex.

<table><tbody><tr><td>1mL syringe</td><td>KOREAVACCINE CORPORATION</td><td></td><td>injecting the drug for anesthesia&amp;nbsp;</td></tr><tr><td>3mL syringe</td><td>KOREAVACCINE CORPORATION</td><td></td><td>injecting the drug for anesthesia&amp;nbsp;</td></tr><tr><td>Bone rongeur</td><td>Fine Science Tools</td><td>16220-14</td><td>remove the skull</td></tr><tr><td>connector</td><td>Gbrain</td><td></td><td>Connect graphene electrode to headstage</td></tr><tr><td>drill</td><td>FALCON tool</td><td></td><td>grind the skull</td></tr><tr><td>drill bits</td><td>Osstem implant</td><td></td><td>grind the skull</td></tr><tr><td>Graefe iris forceps slightly curved serrated</td><td>vubu</td><td>vudu-02-73010</td><td>remove the tissue from the skull or hold wiper</td></tr><tr><td>graphene multielectrode array</td><td>Gbrain</td><td></td><td>records signals from neuron</td></tr><tr><td>isoflurane</td><td>Hana Pharm Corporation</td><td></td><td>sacrifce the subject</td></tr><tr><td>ketamine</td><td>yuhan corporation</td><td></td><td>used for anesthesia</td></tr><tr><td>lidocaine(2%)</td><td>Daihan pharmaceutical&amp;nbsp;</td><td></td><td>local anesthetic</td></tr><tr><td>Matlab R2021b</td><td>Mathworks</td><td></td><td>Data analysis Software</td></tr><tr><td>mosquito hemostats</td><td>Fine Science Tools</td><td>91309-12</td><td>fasten the scalp</td></tr><tr><td>ointment</td><td>Alcon</td><td></td><td>prevent eye from drying out&amp;nbsp;</td></tr><tr><td>povidone</td><td>Green Pharmaceutical corporation</td><td></td><td>disinfect the incision area</td></tr><tr><td>RHS 32ch Stim/Record headstage</td><td>intan technologies</td><td>M4032</td><td>connect connector to interface cable and contain intan RHS stim/amplifier chip</td></tr><tr><td>RHS 6-ft (1.8m) Stim SPI interface cable</td><td>intan technologies</td><td>M3206</td><td>connect graphene electrode to headstage</td></tr><tr><td>RHS Stim/Recording controller software</td><td>intan technologies</td><td></td><td>Data Acquisition Software</td></tr><tr><td>RHS stimulation/ Recording controller</td><td>intan technologies</td><td>M4200</td><td></td></tr><tr><td>saline</td><td>JW Pharmaceutical</td><td></td><td></td></tr><tr><td>scalpel</td><td>Hammacher</td><td>HSB 805-03</td><td></td></tr><tr><td>stereotaxic instrument</td><td>stoelting</td><td></td><td>fasten the subject</td></tr><tr><td>sterile Hypodermic Needle</td><td>KOREAVACCINE CORPORATION</td><td></td><td>remove the dura mater</td></tr><tr><td>Steven Iris Tissue Forceps</td><td>KASCO</td><td>50-2026</td><td>remove the dura mater</td></tr><tr><td>surgical blade no.11</td><td>FEATHER</td><td></td><td>inscise the scalp</td></tr><tr><td>surgical sicssors</td><td>Fine Science Tools</td><td>14090-09</td><td>inscise the scalp and remove the dura mater</td></tr><tr><td>wooden stick</td><td></td><td></td><td>whisker stimulation</td></tr><tr><td>xylazine</td><td>Bayer Korea</td><td></td><td>used for anesthesia</td></tr></tbody></table>

brain mapping using a graphene electrode array

Cortical maps represent the spatial organization of location-dependent neural responses to sensorimotor stimuli in the cerebral cortex, enabling the prediction of physiologically relevant behaviors. Various methods, such as penetrating electrodes, electroencephalography, positron emission tomography, magnetoencephalography, and functional magnetic resonance imaging, have been used to obtain cortical maps. However, these methods are limited by poor spatiotemporal resolution, low signal-to-noise ratio (SNR), high costs, and non-biocompatibility or cause physical damage to the brain. This study proposes a graphene array-based somatosensory mapping method as a feature of electrocorticography that offers superior biocompatibility, high spatiotemporal resolution, desirable SNR, and minimized tissue damage, overcoming the drawbacks of previous methods. This study demonstrated the feasibility of a graphene electrode array for somatosensory mapping in rats. The presented protocol can be applied not only to the somatosensory cortex but also to other cortices such as the auditory, visual, and motor cortex, providing advanced technology for clinical implementation.

This protocol describes how a graphene multichannel array is mounted on the surface of the brain. The somatosensory map was constructed by acquiring neural responses to physical stimuli and calculating the amplitude of the response. Figure 1 shows the schematic of this experiment.
Figure 2A presents the structural characteristics of a graphene electrode array. There are thru-holes of the substrate between the electrodes. These holes help the electrode firmly contact the cortical surface (Figure 2B). The strong adhesion of the electrode to the cortex helps record neural signals with less noise.
Figure 2C (left) shows the location-dependent neural responses acquired by stimulating the whiskers, trunk, paws, and limbs coded in different colors. A rat homunculus, the miniature body of the rat, is drawn with the actual ratio of each color size in the somatosensory cortex map (Figure 2C, right).
Figure 2D presents stimuli-specific responses with colors associated with each body part. The responses are recorded through a graphene electrode array placed on the surface of the cortex. Using the data recorded from the graphene array, the amplitude of SEPs is calculated to obtain the amplitude-dependent somatosensory map.
Sensory stimulus-induced local field potentials enable the construction of the somatosensory map. The response size to each body stimulus poses rodent homunculus. Each color represents a different body part (Figure 3).
The acquired cortex map using this protocol reveals the specific regions within the somatosensory cortex that respond to the whiskers, forepaws, forelimbs, hind paws, hindlimbs, and trunks. It provides insights into the extent of the involvement of the cortical area in processing physical stimulus information for each body part.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64910/64910fig01.jpg" /> 
Figure 1: Schematic of the experiment setup. The graphene-based electrode array is attached to the somatosensory cortex, and the whiskers or other body parts are stimulated by gentle touch. The thick red line represents the cable, and the thin red and blue lines represent the ground and reference wires. The black dot indicates the bregma. The data acquisition system is connected to the computer via USB. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64910/64910fig01large.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/64910/64910fig02.jpg" /> 
Figure 2: Graphene-based microelectrode array for brain mapping on the cortical surface. (A)&#160;Schematic of the graphene-based electrode array. (B)&#160;Optical image of the graphene electrode array on the cortical surface. (C) Rat&#39;s auditory and somatosensory cortices. Two maps of auditory and somatosensory areas responding to auditory stimuli with various frequency tones and physical stimuli applied to each body part. (D) The 30-channel (excluding the reference and ground electrodes) recording of the graphene-electrode array on the cortical surface. Box colors correlate with the geographical locations of the cortical surface. The figures are adapted and modified from Lee et al. (2021).4 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64910/64910fig02large.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/64910/64910fig03.jpg" /> 
Figure 3: Somatosensory map. (A) Location of neural recordings across cortical layers (left). A cortical surface map determined using a graphene electrode array. A color-coded somatosensory map constructed using the response amplitudes and overlapped with the homunculus (right). (B) Recorded cortical SEPs and maps following the stimulation of each body part. This figure is adapted and modified from Lee et al. (2021).4 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64910/64910fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Brain Mapping Using a Graphene Electrode Array at JoVE.com

Brain Mapping Using a Graphene Electrode Array

We present a graphene array-based brain mapping procedure to reduce the invasiveness and improve spatiotemporal resolution. Graphene array-based surface electrodes exhibit long-term biocompatibility, mechanical flexibility, and suitability for brain mapping in a convoluted brain. This protocol allows for constructing multiple forms of sensory maps simultaneously and sequentially.

Cortical maps represent the spatial organization of location-dependent neural responses to sensorimotor stimuli in the cerebral cortex, enabling the ...

brain-mapping-using-a-graphene-electrode-array

Nanyang Technological University

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Neuroscience

1.7K Views.  Incheon National University. We present a graphene array-based brain mapping procedure to reduce the invasiveness and improve spatiotemporal resolution. Graphene array-based surface electrodes exhibit long-term biocompatibility, mechanical flexibility, and suitability for brain mapping in a convoluted brain. This protocol allows for constructing multiple forms of sensory maps simultaneously and sequentially.

Video: Brain Mapping Using a Graphene Electrode Array

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 ...

Development of a 3D Graphene Electrode Dielectrophoretic Device

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The protocol details the procedures to construct a versatile, reusable, multiple layer, laminated dielectrophoresis chamber. Specifically, six layers of 50 &#181;m x&#160;0.7 cm x&#160;2 cm graphene paper and five layers of double sided tape were alternately stacked together, then clamped to a glass slide. Then a 700 &#956;m diameter micro-well was drilled through the laminated structure using a computer-controlled micro drilling machine. Insulating properties of the tape layer between adjacent graphene layers were assured by resistance tests. Silver conductive epoxy connected alternate layers of graphene paper and formed stable connections between the graphene paper and external copper wire electrodes. The finished device was then clamped and sealed to a glass slide. The electric field gradient was modeled within the multi-layer device. Dielectrophoretic behaviors of 6 &#956;m polystyrene beads were demonstrated in the 1 mm deep micro-well, with medium conductivities ranging from 0.0001 S/m to 1.3 S/m, and applied signal frequencies from 100 Hz to 10 MHz. Negative dielectrophoretic responses were observed in three dimensions over most of the conductivity-frequency space and cross-over frequency values are consistent with previously reported literature values. The device did not prevent AC electroosmosis and electrothermal flows, which occurred in the low and high frequency regions, respectively. The graphene paper utilized in this device is versatile and could subsequently function as a biosensor after dielectrophoretic characterizations are complete.

A microdevice with high throughput potential is used to demonstrate three-dimensional (3D) dielectrophoresis (DEP) with novel materials. Graphene nanoplatelet paper and double sided tape were alternately stacked; a 700 &#956;m micro-well was drilled transverse to the layers. DEP behavior of polystyrene beads was demonstrated in the micro-well.

The design and fabrication of a novel 3D electrode microdevice using 50 &#181;m thick graphene paper and 100 &#181;m double sided tape is described. The ...

Engineering

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), ...

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

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

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

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

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Mapping Cerebral Blood Flow and Electrical Fields: Electrode Placement in Mice

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Author Spotlight: Stimulation-Based Approach to Improve Cerebral Blood Flow in Alzheimer's Model 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Microelectrode Array-Based Assessment of Neuronal Networks in Mouse Spinal Cord Slices

Source: Iredale, J. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62920">Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays.</a> J. Vis. Exp. (2022).
This video demonstrates a microelectrode array-based assay for studying neuronal network activity in mouse spinal cord sections. First, the electrophysiological activity from the superficial dorsal horn (SDH) of the slice is recorded. Then, a potassium channel inhibitor is introduced to prolong depolarization, resulting in synchronous rhythmic activity across the neuronal network.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1.&#160;In vitro electrophysiology

	...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Monitoring Seizure-Induced Neural Electrical Activity in Brain Slices Using Microelectrode Arrays

Source: Panuccio, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57548">Recording and modulation of epileptiform activity in rodent brain slices coupled to microelectrode arrays.</a> J. Vis. Exp. (2018)
This protocol involves using a microelectrode array (MEA) to record electrical activity in a brain slice exposed to a seizure-inducing drug. The procedure includes positioning the brain slice in an MEA recording chamber, applying electrical stimulation to induce artificial seizures, and analyzing the resulting signals.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of the MEA ...

Brain Mapping Using a Graphene Electrode Array

Summary

Explore More Videos

Functional Mapping with Simultaneous MEG and EEG

Development of a 3D Graphene Electrode Dielectrophoretic Device

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

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

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Microelectrode Array-Based Assessment of Neuronal Networks in Mouse Spinal Cord Slices

Monitoring Seizure-Induced Neural Electrical Activity in Brain Slices Using Microelectrode Arrays