This method can help answer key questions in the field of neuromodulation for epilepsy treatment, such as what brain area and stimulation protocol are best suited. The main advantage of this technique is that rodent brain slices represent simplified, realistic model of brain circuits while microelectrode arrays allow multiple observation point at known inter-electrode distance. To prepare the MEA, evenly spread the elastomeric sealant on the bottom surface of the recording chamber, and make sure that there are no bubbles.
Next, mount the recording chamber onto the MEA with the help of tweezers, and apply gentle pressure. Spread a sealant layer around the outer border of the recording chamber. Then, place the MEA/chamber assembly in a closed, moist environment, and let it cure at room temperature overnight.
To prepare the recording bench, set the warm bath to 32 degrees Celsius. Next, pour the holding ACSF into the holding and pre-warming chambers. Bubble the solutions with 95%oxygen and 5%carbon dioxide mixture, and remove any trapped bubbles using an inverted Pasteur pipette.
Keep the holding chamber at room temperature, and place the pre-warming chamber in the warm bath. Prepare horizontal hippocampus-cortex brain slices according to standard procedures, and let them recover in the holding chamber at room temperature for at least 60 minutes. Pour 4AP ACSF into an incubating chamber.
Then, bubble it with 95%oxygen and 5%carbon dioxide mixture, and remove any trapped bubbles using an inverted Pasteur pipette. Subsequently, cover the chamber, and transfer it to the warm bath. Make sure that the pre-warming ACSF and the 4AP ACSF temperatures are 30 to 32 degrees Celsius.
Use an inverted glass Pasteur pipette to transfer one brain slice into the pre-warming chamber, and let the tissue rest for 25 to 30 minutes. Then, transfer the brain slice to the 4AP ACSF incubating chamber, and let it rest for 60 minutes. Let the 4AP ACSF flow through the perfusion tubing into a beaker until there is no air inside.
Then, stop the solution flow. Place the dry MEA chip inside the MEA amplifier, and secure the amplifier head. Subsequently, use a plastic Pasteur pipette to transfer 4AP ACSF to the inlet and outlet reservoir of the recording chamber.
Secure the heating cannula to a magnetic holder, and place its tip inside the recording chamber inlet port. Then, attach the magnetic holder to a magnetic strip on the MEA amplifier head. Connect the perfusion tubing to the cannula and the cannula to the thermostat.
Afterward, place the suction needle in the outlet reservoir. Verify that there is negative pressure by submerging the suction needle into the ACSF, and check for a constant low-frequency suction noise. Next, set the flow regulator to allow a flow rate of one milliliter per minute, and start perfusing.
Once the 4AP ACSF is flowing through the cannula, turn on the thermostat. Set the heating cannula to 37 degrees Celsius and the MEA base to 32 degrees Celsius to achieve a temperature of 32 to 34 degrees Celsius inside the recording chamber. Then, place the external reference electrode in the recording chamber inlet reservoir.
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. 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 XY-coordinates for image straightening and electrode mapping. From the slice type drop-down 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 inter-pulse interval of five 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 ictogenesis, 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 pre-stimulus condition by recording at least four ictal discharges. This figure shows the recordings of 4AP-induced ictal activity in the entorhinal cortex and perirhinal cortex during the control condition, periodic pacing at one hertz, and during the recovery upon stimulus withdrawal. This graph shows the quantification of the effect of periodic pacing at one hertz, which indicates a significant reduction of the total seizing time during stimulation.
Once mastered, this technique can be done in three to four hours if it is performed properly. While attempting this procedure, it is important to remember to quickly and correctly place the brain slice onto the MEA. Following this procedure, other methods like pharmacological manipulation can be performed in order to answer additional questions about the contribution of neurotransmitters or ion channel to epileptiform activity and to the effect of neuromodulation.
After its development, this technique paved the way for researcher in the field of neuroscience to explore neuronal network interaction in health and disease in cultured neuron as well in brain slices. After watching this video, you should have a good understanding of how to record and electrically modulate the epileptiform activity induced by four-aminopyridine in rodent brain slices coupled to MEAs.
