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...

<ol>
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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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1mL syringe</td>
			<td>KOREAVACCINE CORPORATION</td>
			<td></td>
			<td>injecting the drug for anesthesia&#160;</td>
		</tr>
		<tr>
			<td>3mL syringe</td>
			<td>KOREAVACCINE CORPORATION</td>
			<td></td>
			<td>injecting the drug for anesthesia&#160;</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&#160;</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&#160;</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 h...

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 ...

The cortical map is a set of local patches representing response properties to sensory motor stimuli in the cerebral cortex. It can discover the spatial formation of neural networks which can offer a prediction for perception and cognition. Hence, cortical maps are used to study neural responses from external stimuli and the processing of sensory motor information To create cortical maps are invasive and non-invasive methods.Using intracortical electrodes is one of the most common invasive methods used for mapping the cerebral cortex. As it can damage the brain for high resolution, it prevents further measurements from being made. Alternative brain mapping tools such as EEG, PET, MEG, fMRI, are non-invasive methods that allow for whole brain mapping and repeated sampling.However, several disadvantages are low spatial temporal resolution, temporal lag, errors due to unspecified modulatory inputs and prohibitive costs. Optical techniques such as calcium imaging and optogenetic fMRI provide large scale brain mapping but are not clinically beneficial due to indicator toxicity and low spatial temporal resolution. The Graphene electrode array exhibits long-term biocompatibility and mechanical flexibility which provides stable recordings of the convoluted brain.Recently, our group has developed a graphene electrode array that provides high resolution recordings for neural signals on the cortical surface. This protocol uses a graphene electrode array to record the SEPs from an SD rat's forepaw, forelimb, hind paw, hind limb, trunk, and whisker to create a cortical map. Administer an intraperitoneal injection of ketamine and xylazine cocktail using a dosage of 90 milligram per kilogram for ketamine and 10 milligram per kilogram for xylazine.To maintain a desired depth of anesthesia throughout the surgery, inject a 45 milligram per kilogram ketamine, and five milligram per kilogram xylazine cocktail if the rat shows signs of awakening. Pinch the toe to confirm the depth of anesthesia. If the rat trembles, wait for additional minutes until it shows no response.Shave the fur on the rat's head from the space between the eyes to the back of the ears using a trimmer. Then apply an ophthalmic ointment onto the eyes. Using ear bars and stereotaxic adaptors, fix the rat's head onto the stereotaxic apparatus.Make sure that the rat's head is fixed properly. Sterilize a shaved area with povidone-soaked cotton swap. Scrub the shaved area with alcohol.Repeat the sterilization process three times. Then inject 0.1 milliliters of 2%lidocaine into the scalp to induce local anesthesia. Creates a two to three-centimeter midline incision and laterally pull apart the scalp To expose the skull.Clamp the scalp to expose the skull with mosquito forceps. Start by removing the periosteum by scratching the surface of the skull with forceps. In a straight line towards the lambda, locate the sternum magna by tearing the muscle behind the occipital bone on the posterior edge.Use a microscope to get a close view of the cisterna magna. Incise the cisterna magna gently. After tearing the cisterna magna, cerebral spinal fluids flow out.Drain the cerebral spinal fluid with the coiled sterile gauze to sink down the brain. Mark where the graphene electrode will be placed based on the predefined stereotaxic coordinates according to the position of the bregma. The somatosensory cortex is located three millimeters in the anterior posterior axis and six millimeters in the right lateral direction from the bregma of the right hemisphere skull.Drill the area marked according to the stereotaxic coordinate. Remove the skull with the bone rongeur. Stick a cotton swab into a 26 garish needle.Bend the needle to 90 degrees. To remove the dura mater, create a hole using a bent needle. Lift the dura mater upward, insert forceps into that hole and proceed to cut the dura mater with scissors or tear it with forceps.Attach the sterile gauze wet with saline on the somatosensory cortex to prevent the cortex from drying out. Detach the graphene multielectrode array without causing any damage by applying saline solution. Remove the outer covering of the reference and ground wires from the connector.To set up the graphene multi electrode array for mapping, connect the head stage with the connector. Connect the graphene electrode array with the connector. Plug the interface cable into the recording controller.Connect the graphene multielectrode array and connector complex to an interface cable. Then secure the graphene multielectrode array complex onto the stereotaxic arm. Place the graphene electrode array onto the somatosensory cortex based on the predefined stereotaxic coordinates.Place the reference wire in the tissue behind the occipital bone. Then connect the ground wire to the grounded optical table. Open the recording software and set the sampling rate at 30 kilohertz and click the OK button.Set the 60 hertz notch filter to remove the noise from power. Bend the whisker with a fine stick. Record neural signals through the data acquisition system for the desired time.These signals are neural signals obtained through stimuli for whiskers. Other body parts also stimulate the same way to record SEPs. Open the MATLAB code named read_Intan_RHS2000 file.Click the Run button. Choose the recording file and click that file and then wait for the file to be read. Enter the command plot to create a 2D line plot.Select the representative peak signals that were in response to the stimulus, and calculate the amplitudes of the SEPs by measuring the maximum and minimum values. Obtain the topographic map by coloring the grade with the different shade of color according to the SEP's amplitude. The graphene electrode array is on the left.There are through holes of the substrate between all electrodes. These holes help the electrodes maintain strong contact with the cortex. On the right, the image shows the electrode array on the cortex.On the left, the ratio of each location-dependent neural response caused by stimulations to different parts of the rat's body is displayed in the shape of a rat. Each body part is represented with a different color. This configures a somatosensory cortex map.On the right, the following figure shows a stimuli specific responses gained through the multichannel graphene electrodes that were placed on the surface of the cortex. Each colored box represents the responses that different body parts had on the 30 channels. The figure above represents the LFPs detected on the surface of the somatosensory cortex by using graphene electrode arrays.The color bar shows that a larger amplitude is represented by a darker shade of color and each color represents a different body part. The figure flex the amplitude based on the shades in the color bar. This homunculus of of the rodent was created using the results that were just explained.The larger the amplitude a certain body parts had, the larger it would be illustrated on the homonculus. The figure below represents a separate data of LFPs collected from each body part. It represents the varying amplitudes of LFPs detected based on the color bar.There are several precautions for this procedure. When removing the cerebral spinal fluid, the experimenter should be careful not to touch the brainstem or a spinal cord. This step takes practice.The rodents whisker is developed enough to sense a deflection direction, stimulus intensity and location of the whisker that was stimulated. That is why the whiskers should be stimulated in a constant direction and intensity for this protocol. Other body parts should also be stimulated consistently.The limitation of this protocol is that signals evoked in deep brain structures can't be recorded as a graphene electrode array is mounted on the cortical surface. Thus, the experimenter can't identify how the columnar network is hierarchically organized concerning the neural responses. Nonetheless, this protocol can be further applied if the experimenter changes the multiple areas where the electrode arrays are placed.For instance, if the experimenter places the electrode on the auditory or visual cortex to extract auditory and visual information, they can acquire the auditory or visual maps. Also, this protocol can be extended to the chronic implantation of the graphene multielectrode array.

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Neuroscience

1.5K vues.  Incheon National University. La carte corticale est un ensemble de patchs locaux représentant les propriétés de réponse aux stimuli sensori-moteurs dans le cortex cérébral. Il peut découvrir la formation spatiale de réseaux neuronaux qui peuvent offrir une prédiction de la perception et de la cognition. Par conséquent, les cartes corticales sont utilisées pour étudier les réponses neuronales des stimuli externes et le traitement de l’information sensori-motrice Pour créer des cartes corticales, les métho...