monitoring seizure-induced neural electrical activity in brain slices using microelectrode arrays

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

Once the 4-AP aCSF level and the recording temperature are stabilized as desired, turn the perfusion and the suction stopcocks to the off position to temporarily stop them. Quickly transfer one brain slice onto the MEA recording chamber using an inverted glass Pasteur pipette.
Adjust its position on the MEA recording area as needed, using a fire-polished curled Pasteur pipette or a soft, compact small brush. Place the hold-down anchor on the brain slice. Restart the perfusion and the suction by turning their stopcocks back to the on position.
Take a picture of the brain slice using a camera mounted on an inverted microscope stage. Then, run the script mapMEA on the computer software to start the GUI to map the electrodes. Click the Browse button to load the picture of the brain slice. Make sure that the reference electrode appears in the upper column of the left half side of the MEA. Then, click the Activate Pointer button.
Select the top and bottom electrodes in the leftmost column of the array to mark the x, y-coordinates for image straightening and electrode mapping. From the Slice Type dropdown menu, select Horizontal. Tick the default structures checkbox.
Using the numbered push buttons below the brain slice picture, select the electrodes corresponding to the ROI, and click the corresponding push buttons in the structure panel to assign them. Repeat this step for each ROI. Afterward, press the Save button. The software will generate a result folder containing a table, reporting the selected electrodes and ROIs.
For electrophysiology study, turn on the stimulus unit at least 10 minutes prior to the stimulation protocol to allow self-calibration and stabilization. Then, start the stimulus control software and verify that the stimulator and MEA amplifier are correctly connected, as indicated by a green LED in the main panel of the stimulus control software.
Set up the stimulation in bipolar configuration. Connect the stimulus unit to the ground. Then, connect the electrode pairs in contact with the pyramidal cell layer of the CA1 proximal subiculum among the ones mapped with the script mapMEA. To acquire data, press the Play button in the main panel of the recording software. Record at least four ictal discharges.
Next, run a fast input/output test to identify the best stimulus intensity. To do so, use the main panel to design a square biphasic positive-negative current pulse with a duration of 100 microseconds per phase.
In the Pulse Amplitude tab, enter an initial pulse amplitude of 100 microamperes per phase. Set the stimulation pulse at 0.2 hertz or lower by adding an interpulse interval of 5 seconds or longer. Increase the stimulus amplitude by 50 to 100-microampere steps at each trial until the stimulation can reliably evoke interictal-like events in the parahippocampal cortices.
For electrical modulation of limbic ectogenesis, program the stimulus unit to deliver the stimulation protocol of interest, and use the stimulus amplitude identified during the input/output test.
After the stimulation stops, verify the network recovery to prestimulus condition by recording at least four ictal discharges.

monitoring-seizure-induced-neural-electrical-activity-brain-slices

Nanyang Technological University

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of the MEA Setup
Note: Start 15 min before recording.
<ol>
	<li>Transfer the remaining volume of 4AP-ACSF (4-aminopyridine artificial cerebrospinal fluid) into a 500 mL Erlenmeyer flask.</li>
	<li>Place the Erlenmeyer flask on a shelf above the MEA (Microelectrode arrays) amplifier and use tubing to allow the solution to continuously feed a 60 mL syringe. Adjust the height of the Erlenmeyer flask and the syringe to allow a gravity-fed rate of 1 mL/min. 
	Note: The correct height depends on the tubing inner diameter (ID). For tubing of 5/32 inches ID, 30 cm is sufficient.</li>
	<li>Start bubbling the 4AP-ACSF in the Erlenmeyer flask and in the syringe with a 95% O2/5% CO2 gas mixture.</li>
	<li>Let the 4AP-ACSF flow through the perfusion tubing into a beaker until there is no air inside; then stop the solution flow.</li>
	<li>Connect the heating element at the MEA base to the thermostat. Place the dry MEA chip inside the MEA amplifier and secure the amplifier head. A plastic Pasteur pipette transfers 4AP-ACSF to the recording chamber&#39;s inlet and outer reservoir.</li>
	<li>Secure the heating cannula to a magnetic holder and place its tip inside the recording chamber inlet port. Attach the magnetic holder to a magnetic strip on the MEA amplifier head. Connect the perfusion tubing to the cannula. Connect the cannula to the thermostat. 
	NOTE: The heating cannula should be covered by beveled polytetrafluoroethylene (PTFE) tubing to reach the recording chamber inlet port and minimize noise due to its metallic material. When the inlet reservoir is adequately filled with 4AP-ACSF, drops falling from the perfusion system should not be visible.</li>
	<li>Place the suction needle inside the reservoir and verify that there is negative pressure by submerging it into the ACSF; check for a constant low-frequency suction noise. 
	Note: The suction needle should be placed so that the 4AP-ACSF flows just above the brain slice surface. A vacuum line or a low-noise vacuum pump can be used.</li>
	<li>Set the flow regulator to allow a flow rate of 1 mL/min and start perfusing. 
	NOTE: Gravity-fed perfusion eliminates the noise that might be caused by peristaltic pumps; if peristaltic pumps are preferred, low-noise models are mandatory.</li>
	<li>Once 4AP-ACSF is flowing through the cannula, turn on the thermostat. Set the heating cannula to 37 &#176;C and the MEA base to 32 &#176;C to achieve a 32 - 34 &#176;C temperature inside the recording chamber. 
	NOTE: CAUTION! Never heat the cannula without solution in it or it might be irreversibly damaged. Set the temperature of the cannula higher than the recording temperature (i.e., 32 - 34 &#176;C) to account for the intrinsic temperature offset between the set value and the actual value at the tip of the heating cannula and within the recording chamber. The flow rate, environment temperature, and volume of the recording chamber all influence the temperature of the recording solution. The settings reported in step 1.9 are optimized for the described protocol and equipment. Always check the actual recording temperature using a thermocouple and adjust the settings as needed. Do not heat the MEA base above 34 &#176;C to avoid overheating the brain slice.</li>
	<li>Place the external reference electrode in the recording chamber inlet reservoir. 
	NOTE: Although MEA chips are equipped with an internal reference electrode, the custom recording chamber covers this. Thus, an external reference electrode must be used. A saturated KCl (Potassium chloride) pellet is the most practical since it is ready to use without the need for chlorination.</li>
</ol>
2. MEA Live Mapping
<ol>
	<li>Once the 4AP-ACSF level and the recording temperature are stabilized as desired, turn the perfusion and the suction stopcocks to the off position to temporarily stop them.
	<ol>
		<li>Quickly transfer one brain slice onto the MEA recording chamber using an inverted glass Pasteur pipette. Adjust its position on the MEA recording area as needed using a fire-polished curled Pasteur pipette (Figure 1C) or a soft, compact brush. Place the hold-down anchor (Figure 1D) on the brain slice. Restart the perfusion and the suction by turning their stopcocks back to the on position. 
		NOTE: CRITICAL! The slice should be transferred, and the perfusion restarted within 60 s, or the tissue might die. The slice hold-down anchor should be kept in 4AP-ACSF to prevent the brain slice from moving while placing the anchor on the brain slice due to differences in superficial tension. The anchor to secure the brain slice onto the MEA can be custom-made using stainless steel wire and nylon thread (Figure 1D) or obtained from commercial sources. Figure 1E shows the final experimental set-up with the MEA chip connected to the amplifier&#39;s head: a brain slice resting on the MEA chip within the recording chamber is held down by the custom anchor. The reference electrode (red circle) and the PTFE tubing covering the heating cannula (red arrow) are positioned in the inlet reservoir, whereas the suction needle (blue arrow) is positioned in the outlet reservoir.</li>
	</ol>
	</li>
	<li>Take a picture of the brain slice using a camera mounted on an inverted microscope stage.</li>
	<li>Run the script mapMEA on the computer software to start the GUI to map the electrodes. 
	NOTE: The custom-made software allows the user to select the electrodes that correspond to specific structures of the brain slice. This step is crucial to activate the correct pathway and suppress ictal activity by using electrical stimulation.</li>
	<li>Click the Browse button to load the picture of the brain slice. Make sure that the reference electrode appears in the upper row of the left half-side of the MEA (Figure 2A, triangle mark). Click the Activate Pointer button, then select the top and bottom electrodes in the leftmost row of the array to mark the XY coordinates for image straightening and electrode mapping.</li>
	<li>From the slice type drop-down menu select Horizontal. Tick the Default structures checkbox. 
	NOTE: It is possible to customize the structures by selecting the Enter New Structures button. The default structures for the horizontal brain slice are depicted in Figure 2A.</li>
	<li>Using the numbered pushbuttons below the brain slice picture, select the electrodes corresponding to the ROI and click the corresponding pushbutton in the structures panel to assign them (Figure 2B); repeat this step for each ROI.</li>
	<li>Press the Save button: the software generates a result folder named #EXP_LabelledElectrodes containing a table reporting the selected electrodes and ROIs.</li>
</ol>
3. Recording and Electrical Modulation of the Epileptiform Activity
<ol>
	<li>Allow the brain slice to stabilize within the recording chamber for 5 - 10 min before recording.</li>
	<li>Turn on the stimulus unit at least 10 min prior to the stimulation protocol to allow self-calibration and stabilization. Start the stimulus control software and verify that the stimulator and MEA amplifier are correctly connected, as indicated by a green LED in the main panel of the stimulus control software.</li>
	<li>Set up the stimulation in a bipolar configuration. Select electrode pairs in contact with the pyramidal cell layer of the CA1/proximal subiculum (cf.24,25) among the ones mapped with the script mapMEA (cf. section 12). Use a wire to connect one of the selected electrodes to the negative plug of the stimulator and the other electrode to the positive plug of the same stimulator channel. Use another wire to connect the ground of the stimulator to the ground of the amplifier.</li>
	<li>Start the recording software. To acquire data, press the PLAY button in the main panel of the recording software. Record at least 4 ictal discharges. 
	NOTE: A sampling frequency of 2 kHz allows acquiring field potentials with fair resolution while minimizing hard disc space usage. A 5-min recording file takes ~80 MB. Higher sampling frequencies may be required, e.g., to record stimulus artifacts or multi-unit activity. To observe field potentials only, use a live low-pass filter at 300 Hz to cut off multi-unit activity.</li>
	<li>Determine the stimulus intensity.
	<ol>
		<li>In the stimulus control software, use the main panel to design a square biphasic positive-negative current pulse duration of 100 &#181;s/phase. 
		NOTE: CAUTION! Direct current stimulation requires a balanced charge pulse to avoid damaging the equipment.</li>
		<li>Run a fast input/output (I/O) test to identify the best stimulus intensity. Deliver the stimulation pulse designed at step 3.5.1 at 0.2 Hz or lower by adding an inter-pulse interval of 5 s or longer in the appropriate form of the stimulus control software. In the pulse amplitude tab enter an initial pulse amplitude of 100 &#181;A/phase and increase by 50 - 100 &#181;A steps at each trial until stimulation can reliably evoke interictal-like events in the parahippocampal cortices (check the signals visualized by the recording software).</li>
	</ol>
	</li>
	<li>Electrical modulation of limbic ictogenesis</li>
	<li>Program the stimulus unit to deliver the stimulation protocol of interest. Use the stimulus amplitude identified during the I/O test. 
	NOTE: The failure rate of evoked responses should be &#8804;20 %.</li>
	<li>After stimulation stops, verify network recovery to pre-stimulus condition by recording at least 4 ictal discharges (as in step 3.4).</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma-Adrich</td>
			<td>P7886</td>
			<td>Needed for MEA coating</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Adrich</td>
			<td>S9888</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Adrich</td>
			<td>P9541</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Adrich</td>
			<td>795488</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>CaCl2*2 H2O</td>
			<td>Sigma-Adrich</td>
			<td>C3306</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma-Adrich</td>
			<td>RDD016</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Adrich</td>
			<td>S5761</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>MgCl2*6 H2O</td>
			<td>Sigma-Adrich</td>
			<td>M2670</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>MgSO4*6 H2O</td>
			<td>Sigma-Adrich</td>
			<td>M5921</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Adrich</td>
			<td>RDD023</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>Pyruvic Acid, 98%</td>
			<td>Sigma-Adrich</td>
			<td>107360</td>
			<td>Chemical</td>
		</tr>
		<tr>
			<td>4-aminopyridine</td>
			<td>Sigma-Adrich</td>
			<td>A78403</td>
			<td>Convulsant drug</td>
		</tr>
		<tr>
			<td>STG-2004</td>
			<td>Multichannel System</td>
			<td>STG4004-1.6mA</td>
			<td>4-channel stimulus generator with voltage (&#177;8 V) and current output (&#177;1.6 mA)</td>
		</tr>
		<tr>
			<td>MEA1060</td>
			<td>Multichannel System</td>
			<td>N/A</td>
			<td>MEA amplifier</td>
		</tr>
		<tr>
			<td>planar MEA</td>
			<td>Multichannel System</td>
			<td>60MEA500/30iR-Ti w/o ring</td>
			<td>Must be without ring to allow using the custom recordingchamber</td>
		</tr>
		<tr>
			<td>McRack</td>
			<td>Multichannel System</td>
			<td></td>
			<td>Recording software</td>
		</tr>
		<tr>
			<td>McStimulus II</td>
			<td>Multichannel System</td>
			<td></td>
			<td>STG4004 control software</td>
		</tr>
		<tr>
			<td>TC02</td>
			<td>Multichannel System</td>
			<td>TC02</td>
			<td>2-channel thermostat</td>
		</tr>
		<tr>
			<td>PH01</td>
			<td>Multichannel System</td>
			<td>PH01</td>
			<td>Heating perfusion canula</td>
		</tr>
		<tr>
			<td>MPH</td>
			<td>Multichannel System</td>
			<td>MPH</td>
			<td>Magnetic holder for PH01 and suction needle</td>
		</tr>
		<tr>
			<td>Elastostil E43</td>
			<td>Wacker</td>
			<td>E43</td>
			<td>Elastomeric sealant used to mount the custom recording chamber onto the MEA</td>
		</tr>
		<tr>
			<td>MEA Custom Chamber</td>
			<td>Crisel Instrument</td>
			<td>SKE-chamber MEA</td>
			<td>Custom recording chamber</td>
		</tr>
		<tr>
			<td>Ag/AgCl electrode, pellet, 1.0 mm</td>
			<td>Crisel Instrument</td>
			<td>64-1309</td>
			<td>Reference electrode for the custom recording chamber</td>
		</tr>
		<tr>
			<td>Tergazyme</td>
			<td>Sigma-Adrich</td>
			<td>Z273287-1EA</td>
			<td>Enzymatic cleaner</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>The Mathworks</td>
			<td></td>
			<td>Programming environment for electrodemapping</td>
		</tr>
		<tr>
			<td>VT1000S</td>
			<td>Leica Biosystems</td>
			<td>VT1000S</td>
			<td>Vibratome</td>
		</tr>
		<tr>
			<td></td>
			<td>Warner Instruments</td>
			<td>64-1309</td>
			<td>Ag-AgCl Electrode Pellet 1.0 mm (E205). Reference electrode.</td>
		</tr>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22762/22762_Figure1.jpg" /> 
Figure 1: Custom equipment used for this protocol. (A) The holding chambers for recovery, pre-warming, and pre-incubation in 4AP are assembled using a beaker and a Petri dish. The Petri dish should be smaller in diameter than the breaker and is held in place by means of a syringe plunger. The bottom of the Petri dish is replaced by a nylon mesh (from soft stockings) glued with cyanoacrylate onto the rim of the Petri dish. Oxygen is supplied via a bent spinal needle (22 G), inserted between the walls of the Petri dish and the beaker. Bubbles should be emerging from the side of the beaker and never reach the nylon mesh to avoid brain slice displacement. The top of a Petri dish can be used as a lid to avoid ACSF evaporation and maintain oxygen saturation. (B) Custom recording chamber. In: inlet reservoir to accommodate the heating cannula and the reference electrode. Out: outlet reservoir to accommodate the suction needle. Rec: recording chamber. (C) A glass Pasteur pipette, curled by fire polishing, is used to handle the tissue and adjust its position within the recording chamber. (D) Custom slice hold-down anchor. (E) Final assembly of the recording chamber mounted onto the MEA chip. A brain slice rests on the bottom, held in place by the anchor. The red arrow indicates the PTFE tubing covering the heating cannula, whereas the red circle indicates the reference electrode, a saturated KCl pellet submerged by ACSF in the inlet reservoir. The blue arrow indicates the suction needle in the outlet reservoir.
<img alt="Figure 2" src="/files/ftp_upload/22762/22762_Figure2.jpg" /> 
Figure 2: GUI for live MEA electrode mapping. (A) Schematic representation of the combined hippocampus-EC slice positioned on the MEA. The default structures used for this protocol are cornu ammonis 3 (CA3), enthorinal cortex (EC), perirhinal cortex (PC), and subiculum (SUB). The reference electrode is schematized as a triangle marker. Electrodes encircled in dotted lines represent the XY coordinates for image straightening. (B) Screen capture of the GUI during the live MEA mapping. The flow chart on the left of the capture indicates the step-by-step procedure to follow.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of the MEA ...

Begin by placing a brain slice on a microelectrodearray or MEA recording chamber filled with artificial cerebrospinal fluid or aCSF containing the test drug.
Adjust the position of the slice and secure it with an anchor.
Start the continuous flow of the aCSF, containing a seizure-inducing test drug.
Capture an image of the slice.
Select the appropriate microelectrodes in contact with the brain slice.
Activate the stimulus unit and connect the wires to the MEA unit.
Record the baseline signals.
Perform a test run to determine the optimal stimulus intensity.
Now, start delivering electrical pulses.
These pulses stimulate the selected brain region, inducing an artificial seizure.
Record the resulting electrical activity to capture seizure-evoked responses.

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Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

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Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

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Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

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

Transcrição

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