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.

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

The authors declare that there are no conflicts of interest associated with this research study.

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 challenges, the protocol emphasizes two critical steps: first, ensuring proper harp orientation during placement to prevent jostling by inflow perfusion (step 4.1.4), and second, using an anti-static wipe to remove excess aCSF around the electrodes, thereby promoting effective slice adhesion (steps 4.1.5-4.1.7). Another common issue addressed in the protocol involves chip hydrophobicity, which can lead to bubble formation and introduce noise during recordings. Steps such as ethanol treatment followed by rinsing with deionized water are recommended to alleviate these issues (steps 3.1.2, 3.1.3, and 3.1.6). However, if the issue persists, there are additional steps that can be taken. Fill the chip well with 140-proof ethanol and use a paintbrush to brush the array lightly for 30 s to 60 s. Remove the ethanol and rinse the chip well three times with deionized water. Afterward, fill the well with aCSF and continue the protocol at step 4.
Maintaining a healthy environment for the brain slice is vital and is managed through the proper placement of the inflow and outflow needles and the perfusion system flow rate (step 4.2.1). It is imperative to properly oxygenate the slice and find the most effective perfusion rate23,51. Due to these issues, interface chambers are commonly used for acute brain slice recordings and provide steadier oxygenation. However, recording with interface chambers has been reported to compress the extracellular space between neurons, which can change the physiology of the slice over time24,52,53. Surface tension dynamics from that altered physiology result in a higher baseline electrical sensitivity of the neurons54. This additional excitability increases the likelihood of seizure-like events and other LFPs of interest. In comparison, submerged chambers, which most CMOS-HD-MEA platforms operate with, can more efficiently deliver drugs and glucose to the slice23,51. To maintain a healthy environment for the slice during a recording session that would last over 2 h with CMOS-HD-MEA platforms, it is beneficial to have another pro-convulsant solution prepared (step 4.2.1.9). This will ensure the slice has enough glucose to continue to produce activity.
The lack of spatial resolution can be major limitations in electrophysiology23,55. For instance, patch-clamping is a technique often used in electrophysiology that can provide great detail about how a single cell behaves during network activity; however, one limitation of patch-clamping is that the recording is performed from a single cell and by default a single location. Patch-clamping can be utilized to study network activity, but it offers a rather limited spatial picture of the network activity41,56. Some imaging techniques do afford broad spatial detail of neuronal activity, such as genetically encoded fluorescent biosensors; however, they do so at the cost of temporal resolution57,58,59. Multimodal approaches, such as single or multichannel electrode recordings, can be combined with calcium imaging to achieve increased spatiotemporal resolution; however, the spatial resolution of the electrophysiological recording is still limited to a few locations60,61. When used properly, CMOS-HD-MEA technology allows users to overcome these limitations by recording with high spatial density (electrodes are typically spaced from 20-200 &#181;m apart) from multiple brain areas simultaneously (Figure 4). CMOS-HD-MEA data can allow researchers to differentiate and characterize the propagating activity patterns across the brain slice with greater detail than most other recording techniques10,62,63. While CMOS-HD-MEA technology exhibits excellent spatial mapping ability with high temporal detail64, there are limitations, such as the inability to record reliably from single cells using cortical slices due to the use of non-penetrating 2D arrays and the inability to be used for in vivo experimentation. CMOS-HD-MEA systems typically record only local field potentials from cortical slices; however, they may capture extracellular action potentials on some channels with high-quality slice preparations, though this capability can be unreliable. Single-cell unit activity can be explored on the recorded channels from the CMOS-HD-MEA system using a high-pass filter (~50 Hz high-pass), combined with spike sorting software65,66,67. It is worth pointing out that it is possible to combine microelectrode recordings with CMOS-HD-MEA systems to possibly achieve single-cell information from a few locations, layered with the rich CMOS-HD-MEA information5. The current method with non-penetrating 2D chips is limited by difficulty in detecting higher frequency activity (150-500 Hz) in cortical brain regions. This is likely due to the distance between the neurons producing the activity and the recording electrodes (Figure 6B). This challenge could be overcome by using new penetrating 3D-CMOS-HD-MEA chips, which record from within the brain slice and not just at its surface68.
Using the above protocol, we generated example data to demonstrate the high-quality LFP signals in the form of seizure-like activity that can be achieved with these instruments (Figure 4 and Figure 5). We demonstrated that seizure-like activity can be observed with different recording paradigms (specifically 4-AP and 0 Mg2+) using CMOS-HD-MEA systems despite idiosyncratic differences between the paradigms.
In summary, the protocol and example data presented using the CMOS-HD-MEA provide a roadmap by which high-fidelity spatial and temporal LFP data can be collected. Future applications of this technique include applying this protocol to additional paradigms that induce epileptiform activity and integrating CMOS-HD-MEA with complementary techniques, like imaging. These studies will undoubtedly aid in our understanding of human seizure disorders using these simple brain slice models69. For example, augmentation of this protocol can provide insightful cross-examination of electrophysiological behavior and genetic mapping from various animal models of epilepsy with mutations in clinically relevant genes70. While we have focused on epileptiform activity, other important network activity, such as long-term potentiation, can also be studied with unprecedented spatial resolution using CMOS-HD-MEA systems. We hope this protocol and technology will be implemented to help others in the field overcome data collection challenges and develop more precise research methods to understand various neurological disorders, such as epilepsy.

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><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

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.

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 and time on the x-axis. In this raster plot, each signal point represents a channel that has exceeded the set LFP voltage threshold (see step 5.2.6 and a previously published work10). Such raster plots indicate when and where electrophysiological activity occurs across space and time (Figure 4A,C,E). Visualizing the data in this manner allows the user to take full advantage of the spatial and temporal resolution of the CMOS-HD-MEA system capabilities. Representative neocortical and hippocampal traces from channels corresponding to the raster plots demonstrate how the specific LFP data for each channel can also be visualized (Figure 4B,D,F). Zoomed-in events of epileptiform discharges are displayed for different brain regions and paradigms in Figure 5, and the comparison of the relative frequency powers we observed from the 0 Mg2+ and the 4-AP paradigms are summarized in Figure 6. Across all displayed frequency bands (1-150 Hz), seizure-like activity in both hippocampal and neocortical regions with both paradigms was significantly greater than baseline, following subtraction of all 60 Hz electrical noise (AC frequency in North America) with a digital notch-filter.
While using either the 0 Mg2+ or the 4-AP paradigm, powerful electrographic seizure-like activity was frequently observed in the neocortical regions. The hippocampal regions displayed more variability between brain slices, with some brain slices demonstrating seizure-like activity and others displaying shorter (~1 s) transient discharges (Figure 4 and Figure 5). The power for each recording was normalized to a 10 s &#34;quiet&#34; baseline section at the beginning of each recording in which no discharges were observed. All further discussion of &#34;power&#34; denotes spectral power compared to an in-sample control baseline. Neocortical activity was found to be more powerful in multiple frequency bands under the 0 Mg2+ paradigm when compared to the 4-AP paradigm. This was true for the delta (1-4 Hz), theta (4-8 Hz), low gamma (30-60 Hz) and high gamma (60-150 Hz) frequency bands (Figure 6). In addition, the hippocampal activity in the 0 Mg2+ paradigm demonstrated higher power in the high gamma frequency bands (60-150 Hz) when compared to the neocortex in the 0 Mg2+ paradigm and when also compared to both brain regions assayed in the 4-AP paradigm (Figure 6). Although the 4-AP paradigm exhibited less power in most frequency bands in our studies, compared to the 0 Mg2+ conditions, the hippocampal 4-AP samples tended to demonstrate more overall epileptiform discharges (Figure 4D). We did not observe any difference between brain regions or paradigms when analyzing high-frequency activity (Figure 6B). This is likely because the recordings are made on a 2D, non-penetrating recording chip, meaning that high-frequency activity might not be picked up due to the distance between the neurons producing the activity and the electrode recording sites. Taken together, the results presented here suggest that when using the CMOS-HD-MEA systems with 2D CMOS-MEA chips, one will generally achieve greater overall power during epileptiform discharges using the 0 Mg2+ paradigm, where quality data can be recorded up to the high gamma bands (from 1 up to 150 Hz). In addition, the data presented here suggests that the 4-AP model might not be a great choice with this system if one desires to study information from the gamma bands using this protocol. However, it provides rich data in the lower power bands while also displaying many epileptiform discharges. Importantly, both models will display high-quality seizure-like activity with the above protocol.
Differences in the seizure models likely result in characteristically unique patterns of induced seizure-like activity based on the cellular mechanisms of these paradigms and due to brain region microanatomy41. For example, 4-AP blocks certain classes of voltage-gated K+ channels (primarily the Kv3 family), extending the duration of neuronal excitability and resulting in an onset of epileptiform activity within a few minutes of exposure within the hippocampus42,43,44,45,46. However, the neocortex typically has a much longer delayed onset to epileptiform activity than the hippocampus when exposed to 4-AP41. On the other hand, the 0 Mg2+ paradigm induces epileptiform activity primarily by washing out the Mg2+ block of NMDA receptors in glutamatergic neurons, resulting in early neocortical epileptiform activity47,48,49,50. Although there were significant differences across brain regions and paradigms for many of the highlighted groups, some key similarities between the paradigms and brain regions also remained. The power in both alpha and beta frequency bands were comparable across brain regions and paradigms, and within hippocampal regions, the paradigms showed similar performance in the delta band (Figure 6). Additionally, an overall increase in power compared to baseline for all frequencies (1-150 Hz) was observed during epileptiform activity (Figure 5 and Figure 6), as expected. A final noteworthy observation of the neocortical areas during exposure to 4-AP was the striated pattern observed in the spectrograms depicting greater activity for higher frequencies during ictal start followed by a gradual decrease in frequency as the activity progressed (Figure 5C), possibly suggesting high interneuronal synchrony during the ictal start.

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

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.

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 ...

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

Nanyang Technological University

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Neuroscience

2.2K Views.  Brigham Young University. In het Parrish-lab willen we graag begrijpen hoe aanvallen beginnen, zich verspreiden en eindigen. We zijn vooral geïnteresseerd in het onderzoeken van nieuwe therapieën voor status epilepticus, een levensbedreigende aandoening waarbij een aanval niet vanzelf eindigt. In ons onderzoek gebruiken we CMOS-micro-elektrode-arraysystemen met hoge dichtheid.Deze geavanceerde technologieën stellen ons in staat om elektrofysiologische gegevens met hoge resol...

Video: Auteur in de schijnwerpers: het ontrafelen van de dynamiek van aanvallen en nieuwe therapieën voor status epilepticus met behulp van CMOS-micro-elektrode-arraysyst

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

High-density EEG Recordings of the Freely Moving Mice using Polyimide-based Microelectrode

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a noninvasive brain monitoring tool in cognitive neuroscience as well as a diagnostic tool for epilepsy and sleep disorders in neurology. However, the underlying mechanism of EEG rhythm generation is still under the veil. Recently introduced polyimide-based microelectrode (PBM-array) for high resolution mouse EEG1 is one of the trials to answer the neurophysiological questions on EEG signals based on a rich genetic resource that the mouse model contains for the analysis of complex EEG generation process. This application of nanofabricated PBM-array to mouse skull is an efficient tool for collecting large-scale brain activity of transgenic mice and accommodates to identify the neural correlates to certain EEG rhythms in conjunction with behavior. However its ultra-thin thickness and bifurcated structure cause a trouble in handling and implantation of PBM-array. In the presented video, the preparation and surgery steps for the implantation of PBM-array on a mouse skull are described step by step. Handling and surgery tips to help researchers succeed in implantation are also provided.

In this article, we described the surgery procedure and handling tips for implantation of ultra-thin polyimide-based microelectrode array (PBM-array) on the mouse skull for acquisition of high-density encephalography (EEG) in a mouse model.

Electroencephalogram (EEG) indicates the averaged electrical activity of the neuronal populations on a large-scale level. It is widely utilized as a ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Medicine

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While these systems can provide a wealth of behavioral information, the cost and complexity of these systems can be prohibitive for many labs. We have developed a low-cost assay for measuring locomotive behavior and seizure movement in D. melanogaster. The system uses a web-cam to capture images that can be processed using a combination of inexpensive and free software to track the distance moved, the average velocity of movement and the duration of movement during a specified time-span. To demonstrate the utility of this system, we examined a group of D. melanogaster mutants, the Bang-sensitive (BS) paralytics, which are 3-10 times more susceptible to seizure-like activity (SLA) than wild type flies. Using this novel system, we were able to detect that the BS mutant bang senseless (bss) exhibits lower levels of exploratory locomotion in a novel environment than wild type flies. In addition, the system was used to identify that the drug metformin, which is commonly used to treat type II diabetes, reduces the intensity of SLA in the BS mutants.

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Using a webcam and a combination of free and inexpensive software programs, locomotive patterns in Drosophila melanogaster can be analyzed to detect differences in the speed, distance, and time of various motor activities.

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

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) ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

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

Source: Panuccio, G., et al. <a href="https://app.jove.com/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 ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Monitoring the Brain Cortex with a Multi-Electrode Array in Response to Seizure-Inducing Agents

Source: Lu, H. C., et al. <a href="https://app.jove.com/v/53709">Direct-current stimulation and multi-electrode array recording of seizure-like activity in mice brain slice preparation.</a> J. Vis. Exp. (2016).
In this video, the effects of transcranial direct-current stimulation (tDCS) on brain slices containing the anterior cingulate cortex (ACC) and thalamus regions are investigated, using a multi-electrode array (MEA) to measure electric signals. By delivering electric pulses and infusing seizure-inducing drugs, the study investigates the ACC response to seizures.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparing Experimental ...

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

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