<meta charset="utf8"></head><body>אינדוקציה ורישום עקביים של פעילות דמוית התקף באמצעות CMOS-HD-MEAs

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2D Workbench</td>
			<td>Cloudray</td>
			<td>LM04CLLD26B</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Aminopyridine</td>
			<td>Sigma-Aldrich</td>
			<td>275875</td>
			<td></td>
		</tr>
		<tr>
			<td>Accura Chip</td>
			<td>3Brain</td>
			<td>Accura HD-MEA</td>
			<td>CMOS-HD-MEA chip</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP160-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Kinetic Systems</td>
			<td>91010124</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker for the slice holding chamber, 270 mL</td>
			<td>VWR</td>
			<td>10754-772</td>
			<td></td>
		</tr>
		<tr>
			<td>BioCam</td>
			<td>3Brain</td>
			<td>BioCAM DupleX</td>
			<td>CMOS-HD-MEA platform</td>
		</tr>
		<tr>
			<td>Brainwave Software</td>
			<td>3Brain</td>
			<td>Version 4</td>
			<td>CMOS-HD-MEA software</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP510-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>X02OX95C2003102</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>12005</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen Stones</td>
			<td>Supelco</td>
			<td>59277</td>
			<td></td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precissionary</td>
			<td>VF-300-0Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell</td>
			<td>Precission3650</td>
			<td></td>
		</tr>
		<tr>
			<td>Crocodile Clip Grounding Cables</td>
			<td>JWQIDI</td>
			<td>B06WGZG17W</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent</td>
			<td>Metrex</td>
			<td>10-4100-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Macron Fine Chemicals</td>
			<td>4912-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dihydrogen Sodium Phosphate</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP329-500</td>
			<td></td>
		</tr>
		<tr>
			<td>DinoCam</td>
			<td>Dino-Lite</td>
			<td>AM73915MZTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>A407P-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11980-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>SP88857200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice Machine</td>
			<td>Hoshizaki</td>
			<td>F801MWH</td>
			<td></td>
		</tr>
		<tr>
			<td>Inflow and outflow needles</td>
			<td>Jensen Global</td>
			<td>JG 18-3.0X</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorine</td>
			<td>Dechra</td>
			<td>08PB-STE22002-0122</td>
			<td></td>
		</tr>
		<tr>
			<td>Kim Wipes</td>
			<td>Thermo Fisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>FLM33500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipets</td>
			<td>Gilson</td>
			<td>F144069</td>
			<td></td>
		</tr>
		<tr>
			<td>Mili-Q Water Filter</td>
			<td>Mili-Q</td>
			<td>ZR0Q008WW</td>
			<td></td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Daler Rowney</td>
			<td>AF85 Round: 0</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper Filter</td>
			<td>Whatman</td>
			<td>EW-06648-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>American National Can</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion System</td>
			<td>Multi Channel System</td>
			<td>PPS2</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetor</td>
			<td>Thermo Fisher Scientific</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Harp</td>
			<td>3Brain</td>
			<td>3Brain</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>P330-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Personna</td>
			<td>BP9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Metter Toledo</td>
			<td>AB204</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Solingen</td>
			<td>92008</td>
			<td></td>
		</tr>
		<tr>
			<td>Slice Holding Chamber</td>
			<td>Custom</td>
			<td>Custom</td>
			<td>Custom 3D Printer Design, available upon request</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Macron Fine Chemicals</td>
			<td>7412-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Control Box</td>
			<td>Warner Instruments</td>
			<td>TC344B</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Pipettes</td>
			<td>Genesee Scientific</td>
			<td>30-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>Tygon</td>
			<td>B-44-3 TPE</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome VZ-300</td>
			<td>Precissionary</td>
			<td>VF-00-VM-NC</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Boat</td>
			<td>Electron Microscopy Sciences</td>
			<td>70040</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-quality seizure-like activity from acute brain slices using a complementary metal-oxide-semiconductor high-density microelectrode array system

Commercially available high-density microelectrode array (HD-MEA) systems, which include an MEA chip with thousands of recording points1,2 and an MEA platform to amplify and digitize the data, are an emerging tool for electrophysiological research. These HD-MEA systems use complementary metal-oxide-semiconductor (CMOS) technology to record electrophysiological data with high sensitivity from cell cultures and ex vivo brain slice preparations. These MEA systems afford unprecedented spatial and temporal resolution to neurophysiological research via high electrode density and quality signal-to-noise ratios3. This technology has mostly been used to study extracellular action potentials, but it can also capture high-quality local field potentials (LFPs) from various neuronal brain slice preparations4,5,6,7,8,9,10,11,12,13,14,15. Due to the above-mentioned high-resolution recording capability of CMOS-HD-MEA systems, users can track electrophysiological activity with great spatial accuracy16,17,18. This capability is particularly relevant to tracking propagation patterns of network LFPs5,12,15,19,20,21. Therefore, CMOS-HD-MEA systems can provide an unprecedented understanding of the propagation patterns of physiological and pathological activity from various cell culture and brain slice preparations. Of particular note, these capabilities of CMOS-HD-MEA systems can allow researchers to contrast seizure patterns of different brain regions simultaneously and assay how various anti-epileptic compounds affect these patterns. By doing so, it provides an innovative method for studying ictogenesis and ictal propagation and for understanding how pharmacology disrupts pathological network activity7,10,14. Therefore, these novel capacities of CMOS-HD-MEA systems can contribute significantly to the research of neurological disorders, as well as aid in drug discovery research5,7,11,22. We aim to provide details on using CMOS-HD-MEA systems to study seizure-like activity.
When using CMOS-HD-MEA systems to study LFPs, such as epileptiform activity in acute brain slices, users can face many challenges, including debilitating electrical noise, keeping the slice healthy during experimentation, and detecting a quality signal from a two-dimensional (2D) CMOS-MEA chip that records only from the surface of the brain slice. This protocol describes basic steps for properly grounding the MEA platform and other equipment used in experimentation, a crucial step that may require individual customization for each lab setup. In addition, we discuss steps to aid in keeping the brain slice healthy during long recordings in the submerged chambers used with CMOS-HD-MEA systems23,24,25. Additionally, in contrast to more common electrophysiological recording methods, which record from deep in the brain slice, most CMOS-HD-MEA systems use 2D chips that do not penetrate into the slice. Therefore, these systems require a healthy neuronal outer layer to produce the majority of the recorded LFP signals. Other challenges include visualizing the massive amount of data generated by thousands of electrodes. To overcome these challenges, we recommend a simple but effective protocol that increases the likelihood of achieving high-quality network epileptiform activity that propagates across the brain slice. We also include a brief description of a publicly available graphic user interface (GUI) we developed with associated resources to aid in data visualization10.
Previous publications have provided related protocols for the use of MEA recording systems26,27,28,29. However, this work aims to assist experimenters using CMOS-HD-MEA systems with 2D chips, specifically those seeking to study high-quality epileptiform activity from brain slices. In addition, we compare two of the most common solution manipulations for induction of seizure-like activity, namely the 0 Mg2+ and 4-AP paradigms, to help users identify the most appropriate convulsant media for their specific application. Although the protocol is focused primarily on the generation of seizure-like activity, it can be modified to explore other electrophysiological phenomena using brain slices.

<meta charset="utf8"></head><body>מחבר זרקור: חשיפת דינמיקת התקפים וטיפולים חדשניים לסטטוס אפילפטיקוס באמצעות מערכות מערך מיקרואלקטרודות בצפיפות גבוהה CMOS

פעילות דמוית פרכוסית באיכות גבוהה מפרוסות מוח חריפות באמצעות מערכת משלימה של מערך מיקרואלקטרודות בצפיפות גבוהה של מתכת-תחמוצת-מוליכים למחצה

In the Parrish lab, we are keen on understanding how seizures start, propagate, and terminate. We are particularly interested in exploring novel therapies for status epilepticus, a life-threatening condition in which a seizure does not self-terminate. We use CMOS high-density microelectrode array systems in our research.These advanced technologies allow us to record high resolution electrophysiological data from brain slices, capturing detailed local field potentials. This helps us understand complex brain activities, like seizure patterns, with great spatial and temporal precision. In the future, we plan to explore the spatial and temporal propagation patterns of status epilepticus, a prolonged seizure state that often becomes resistant to anti-epileptic medication.We plan to use the information from these studies to find more effective treatments for status epilepticus.

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

high-quality-seizure-like-activity-from-acute-brain-slices-using

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Research

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

JoVE Journal

Neuroscience

1.3K Views.  Brigham Young University. במעבדת פאריש, אנו מעוניינים להבין כיצד התקפים מתחילים, מתפשטים ומסתיימים. אנו מתעניינים במיוחד בחקר טיפולים חדשניים לסטטוס אפילפטיקוס, מצב מסכן חיים שבו התקף אינו מסיים את עצמו. אנו משתמשים במערכות מערך מיקרואלקטרודות CMOS בצפיפות גבוהה במחקר שלנו.טכנ...

Video: מחבר זרקור: חשיפת דינמיקת התקפים וטיפולים חדשניים לסטטוס אפילפטיקוס באמצעות מערכות מערך מיקרואלקטרודות בצפיפות גבוהה CMOS

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures and ex vivo brain slices in unprecedented electrophysiological detail. CMOS-HD-MEAs were first optimized to record high-quality neuronal unit activity from cell cultures but have also been shown to produce quality data from acute retinal and cerebellar slices. Researchers have recently used CMOS-HD-MEAs to record local field potentials (LFPs) from acute, cortical rodent brain slices. One LFP of interest is seizure-like activity. While many users have produced brief, spontaneous epileptiform discharges using CMOS-HD-MEAs, few users reliably produce quality seizure-like activity. Many factors may contribute to this difficulty, including electrical noise, the inconsistent nature of producing seizure-like activity when using submerged recording chambers, and the limitation that 2D CMOS-MEA chips only record from the surface of the brain slice. The techniques detailed in this protocol should enable users to consistently induce and record high-quality seizure-like activity from acute brain slices with a CMOS-HD-MEA system. In addition, this protocol outlines the proper treatment of CMOS-HD-MEA chips, the management of solutions and brain slices during experimentation, and equipment maintenance.

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain slices.

Equipment Preparation for CMOS-HD-MEA Experimentation

Before starting chip preparation, designate transfer pipettes for various tasks. Fill the well of the micro electrode array or MEA chip with 190 proof ethanol so that the bottom of the chip well is completely covered. Let the ethanol sit for 30 to 60 seconds, then remove it with a discard pipette.Fill the well of the MEA chip with aCSF and remove it with a discard pipette to rinse residual ethanol out of the chip well. Then, add aCSF, and let it stand for at least 30 seconds. Before docking the MEA chip, wet an anti-static wipe with 190 proof ethanol, and use it to wipe the chip's pins.Gently slide the MEA chip into the MEA platform and engage the docking mechanism to lock the chip into place. Check the recording and reference electrodes for bubbles. If bubbles are present, take a clean paintbrush, and lightly sweep over the electrodes to remove them.Check the chip for noise using the CMOS HD-MEA software, and visually scan the false color map for bubbles, non-biological oscillations, or spikes caused by electrical interference. Ground the MEA system appropriately to negate any encountered noise.

הכנת ציוד לניסויי CMOS-HD-MEA

Chip Preparation and Placement of Brain Slice to Record High-Quality Seizure-like Activity Using CMOS-HD-MEA System 

To begin, place a platinum harp in a weighboat near the MEA platform. Then cover the harp with about three milliliters of aCSF to reduce its hydrophobic tendencies. Use scissors to trim about 1.5 inches off the narrow tip of a transfer pipette.Next, use the modified pipette to collect a brain slice from the slice-holding chamber. Gently dispense the brain slice and any solution in the pipette into the chip well. To position the slice properly, use a soft paintbrush to create a current in the solution that pushes the brain slice onto recording electrodes.Using forceps, gently place the harp over the brain slice with the threads downwards to press the slice onto the recording electrodes. Orient the harp so that the side without a frame faces toward the inflow needle and the frame of the harp does not contact any of the recording electrodes. Now take a discard pipette and remove excess aCSF.Then take an antistatic wipe, twist a corner to create a tip, and use it to soak up the remaining aCSF surrounding the recording electrodes without touching the recording electrodes, brain slice, or harp. Using a designated aCSF pipette, quickly add about two milliliters of carbogenated aCSF to cover the brain slice. Fill the well with about three milliliters carbogenated aCSF until it is roughly three quarters full.Then use a microscope or camera to take a high-resolution picture of the brain slice on the MEA chip. Place the inflow and outflow tubes into the beaker filled with aCSF. Place the inflow needle close to the bottom of the chip well, just outside the recording electrodes.Then place the outflow needle close to the top of the chip well, towards the edge, so that the liquid rises almost to the brim of the chip well, about four milliliters. Now set the perfusion inflow to five milliliters per minute and the perfusion outflow to seven milliliters per minute. Turn on the inflow and outflow.Remove the inflow needle from the chip well until it begins to output solution instead of air. After that, place the needle back inside the chip well. Then use a solution heater to keep the solution at or near physiological temperature, around 34 to 37 degrees Celsius.Let the aCSF perfuse over the brain slice for 10 minutes. After 10 minutes have elapsed, move the outflow tube to the discard beaker. Then move the inflow tube to the beaker containing the pro-convulsant solution.Allow the non-convulsant aCSF to be flushed out of the perfusion system into the discard beaker for 10 minutes. Finally, transfer the outflow tube into the beaker containing the pro-convulsant solution and allow it to cycle until the experiment finishes. Powerful electrographic seizure-like activity was frequently observed in the neocortical regions under both the zero magnesium and 4-aminopyridine paradigms.The hippocampal regions displayed more variability between brain slices, with some demonstrating seizure-like activity and others showing transient discharges. Neocortical activity under the zero magnesium paradigm showed higher power in multiple frequency bands compared to the 4-aminopyridine paradigm. Hippocampal activity in the zero magnesium paradigm demonstrated higher power in high gamma frequency bands compared to the neocortex and both brain regions in the 4-aminopyridine paradigm.