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

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.

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.