The authors thank former and current Parrish lab members for their edits on this manuscript. We would also like to thank Alessandro Maccione of 3Brain for his feedback on this work. This work was funded by an AES/EF Junior Investigator Award and by Brigham Young University Colleges of Life Sciences and of Physical and Mathematical Sciences.

Procedures involving mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Brigham Young University. Male and female (n = 8) C57BL/6 mice aged to at least P21 were used in the following experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67065/67065fig01.jpg" /> 
Figure 1: Schematic figure of CMOS-HD-MEA experimentation. (A) The brain sl...

Consistent Induction and Recording of Seizure-Like Activity Using CMOS-HD-MEAs

This protocol includes specific guidelines related to acute brain slice management that address common problems faced by CMOS-HD-MEA users, namely noise development under the brain slice and maintaining a healthy environment for the brain slice. Noise development under the slice occurs when the slice is not properly adhered to the array; if the brain slice is not adequately adhered, air pockets can form underneath the slice, which results in noise. This will result in the inability to acquire data. To mitigate these chal...

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.

<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>
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		<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

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.

Author Spotlight: Unraveling Seizure Dynamics and Novel Therapeutics for Status Epilepticus Using CMOS High-Density Microelectrode Array Systems

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

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.

As is standard when visualizing activity from many channels1,4,5,10, we find it beneficial to first generate a raster plot of the data we acquire with the CMOS-HD-MEA (Figure 4A,C,E). This plot can create a bird&#39;s-eye view of the activity in all the recording channels in each brain slice by displaying each channel on the y-axis...

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.

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