A subscription to JoVE is required to view this content. Sign in or start your free trial.
* These authors contributed equally
Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs) 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 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.
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.
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.
Figure 1: Schematic figure of CMOS-HD-MEA experimentation. (A) The brain slice is prepared by one's preferred cutting method and sub-dissected to fit on the MEA. (B) Prepare the solutions and the CMOS-HD-MEA chip. (C) The sub-dissected brain slice is placed on the electrode array and bathed in the appropriate solutions. (D) Relevant channels are selected from the collected data. Data is then prepared for analysis in the preferred program of the user. Please click here to view a larger version of this figure.
Name | Concentration (mM) | g/L | ||
Sodium Chloride (NaCl) | 126 | 7.36 | ||
Potassium Chloride (KCl) | 3.5 | 0.261 | ||
Dihydrogen Sodium Phosphate (NaH2PO4) | 1.26 | 0.151 | ||
Sodium Bicarbonate (NaHCO3) | 26 | 2.18 | ||
Glucose (C6H12O6) | 10 | 1.80 | ||
Magnesium Chloride (MgCl2) | 1 (from 1 M stock) | 1 mL | ||
Calcium Chloride (CaCl2) | 2 (from 1 M stock) | 2 mL |
Table 1: aCSF solution.
1. Preparing solutions
2. Preparing rodent brain slices
3. Equipment preparation
Figure 2: Configuration and technology diagrams. (A) Diagram of the selection of acute mouse brain slices used in the experiment highlighted by this protocol. (1) Hippocampal region (2) Neocortex region. (B) The proper placement of an acute mouse brain slice and harp on the microelectrode Array (MEA). (C) The anatomy of a 3Brain Accura CMOS-HD-MEA chip. (D) The proper configuration of perfusion inlets and outlets. Input should be deep in the chip well, whereas the output should be on the opposite side of the inlet at the top of the chip well to guarantee a constant flow of fresh, oxygenated aCSF. Please click here to view a larger version of this figure.
Figure 3: Schematic diagram of chip preparation and placement for brain slice experiments. (A) Rinse the chip well with ethanol once, then aCSF three times. (B) Wipe the pins with ethanol using an anti-static wipe. (C) Dock the chip. (D) Place the brain slice on the electrodes. (E) Place the harp on the brain slice (see Figure 2 for proper placement guidelines). (F) Dab the corner of the recording electrodes' well near the brain slice with a twisted anti-static wipe. Please click here to view a larger version of this figure.
4. Experimentation
5. Data analysis
NOTE: There are a variety of analysis packages used for analyzing electrophysiological data produced by CMOS-HD-MEAs, including BrainWave from 3Brain, Yet Another Spike Sorter (YASS), and custom Python tools34,35,36,37. We extracted data from the BrainWave data file format for use in the Xenon LFP Analysis platform to generate the data presented in Figure 4 and Figure 5. Custom Matlab code was used to analyze the data in Figure 6. Protocols for the Xenon LFP Analysis platform are publicly available10. The following protocol steps are specific to recordings made with Brainwave 438; for other systems, refer to supporting documentation related to those systems34,35,36,37. An overview of the analysis steps taken to produce the data with this protocol is provided below. For complete details of how to export, visualize, and analyze data, including tutorial videos and all relevant code files, see39.
Figure 4: Example of evolving epileptiform activity from the 0 Mg2+ and 4-AP paradigms. (A) Example raster plot from the application of aCSF with 0 Mg2+ over approximately 40 min. (B) Example electrophysiology traces taken from the neocortex (blue) and hippocampus (red) demonstrating epileptiform activity from the 0 Mg2+ paradigm. (C) Example raster plot from the application 100 mM 4-AP over approximately 40 min. (D) Example electrophysiology traces taken from the neocortex (purple) and hippocampus (green) demonstrating epileptiform activity from the application of 4-AP. (E) Example raster plot from the application of aCSF with 0 Mg2+ over approximately 40 minutes showing bursting activity as opposed to seizure-like activity as found in the other representative traces. (F) Example electrophysiology traces taken from the neocortex (dark purple) and hippocampus (rust) demonstrating sub-optimal activity from the 0 Mg2+ paradigm intended for comparison to the quality seizure-like activity found in B and D. Please click here to view a larger version of this figure.
Figure 5: Representative results of epileptiform discharges from both the 0 Mg2+ and 4-AP paradigms. (A) Example plots of a typical neocortical seizure-like event induced by the 0 Mg2+ paradigm including (Ai) a spectrogram from a seizure-like event, (Aii) the associated electrophysiological trace, (Aiii) an 80 Hz high pass filter applied to the trace from Aii, (Aiiii) and a magnified section of the trace from Aii. (B) Example plots of a typical hippocampal epileptiform burst induced by the 0 Mg2+ paradigm including (Bi) a spectrogram of the epileptiform burst, (Bii) the associated electrophysiological trace, (Biii) an 80 Hz high pass filter applied to the trace from Bii, (Biiii) and a magnified section of the trace from Bii (C) Example plots of a typical neocortical seizure-like event induced bythe 4-AP paradigm including (Ci) a spectrogram of epileptiform activity, (Cii) the associated electrophysiological trace, (Ciii) an 80 Hz high pass filter applied to the trace from Cii, (Ciiii) and a magnified section of the trace from Cii (D) Example plots of a hippocampal epileptiform burst under the 4-AP paradigm including (Di) a spectrogram of epileptiform activity, (Dii) the associated electrophysiological trace, (Diii) an 80 Hz high pass filter applied to the trace from Dii, (Diiii) and a magnified section of the trace from Dii. Please click here to view a larger version of this figure.
Figure 6: Comparison of percent of baseline power in the various bands across paradigm and brain region during stereotypical epileptiform discharges. (A) The power during epileptiform discharges was significantly different between the paradigms and brain regions for most frequency bands (2-way ANOVA with Tukey test, *P < 0.05, **P < 0.001, ***P < 0.0001). The middle line for each box represents the mean, the borders of the box ±1 standard error of the mean (SEM), and the outermost lines ±2 SEM. (B) Both paradigms and brain regions demonstrated limited power in bands related to high-frequency activity above 150 Hz. Please click here to view a larger version of this figure.
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's-eye view of the activity in all the recording channels in each brain slice by displaying each channel on the y-axis...
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...
The authors declare that there are no conflicts of interest associated with this research study.
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.
Name | Company | Catalog Number | Comments |
2D Workbench | Cloudray | LM04CLLD26B | |
4-Aminopyridine | Sigma-Aldrich | 275875 | |
Accura Chip | 3Brain | Accura HD-MEA | CMOS-HD-MEA chip |
Agarose | Thermo Fisher Scientific | BP160-100 | |
Vibration isolation table | Kinetic Systems | 91010124 | |
Beaker for the slice holding chamber, 270 mL | VWR | 10754-772 | |
BioCam | 3Brain | BioCAM DupleX | CMOS-HD-MEA platform |
Brainwave Software | 3Brain | Version 4 | CMOS-HD-MEA software |
Calcium Chloride | Thermo Fisher Scientific | BP510-500 | |
Carbogen | Airgas | X02OX95C2003102 | |
Carbogen | Airgas | 12005 | |
Carbogen Stones | Supelco | 59277 | |
Compresstome | Precissionary | VF-300-0Z | |
Computer | Dell | Precission3650 | |
Crocodile Clip Grounding Cables | JWQIDI | B06WGZG17W | |
Detergent | Metrex | 10-4100-0000 | |
D-Glucose | Macron Fine Chemicals | 4912-12 | |
Dihydrogen Sodium Phosphate | Thermo Fisher Scientific | BP329-500 | |
DinoCam | Dino-Lite | AM73915MZTL | |
Ethanol | Thermo Fisher Scientific | A407P-4 | |
Forceps | Fine Science Tools | 11980-13 | |
Hot plate | Thermo Fisher Scientific | SP88857200 | |
Ice Machine | Hoshizaki | F801MWH | |
Inflow and outflow needles | Jensen Global | JG 18-3.0X | |
Inline Solution Heater | Warner Instruments | SH-27B | |
Isofluorine | Dechra | 08PB-STE22002-0122 | |
Kim Wipes | Thermo Fisher Scientific | 06-666 | |
Magnesium Chloride | Thermo Fisher Scientific | FLM33500 | |
Micropipets | Gilson | F144069 | |
Mili-Q Water Filter | Mili-Q | ZR0Q008WW | |
Paintbrush | Daler Rowney | AF85 Round: 0 | |
Paper Filter | Whatman | EW-06648-24 | |
Parafilm | American National Can | PM996 | |
Perfusion System | Multi Channel System | PPS2 | |
Pipetor | Thermo Fisher Scientific | FB14955202 | |
Platinum Harp | 3Brain | 3Brain | |
Potassium Chloride | Thermo Fisher Scientific | P330-3 | |
Razor blade | Personna | BP9020 | |
Scale | Metter Toledo | AB204 | |
Scissors | Solingen | 92008 | |
Slice Holding Chamber | Custom | Custom | Custom 3D Printer Design, available upon request |
Sodium Bicarbonate | Macron Fine Chemicals | 7412-06 | |
Sodium Chloride | Thermo Fisher Scientific | S271-3 | |
Temperature Control Box | Warner Instruments | TC344B | |
Transfer Pipettes | Genesee Scientific | 30-200 | |
Tubing | Tygon | B-44-3 TPE | |
Vibratome VZ-300 | Precissionary | VF-00-VM-NC | |
Weigh Boat | Electron Microscopy Sciences | 70040 |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved