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.
- Transfer the remaining volume of 4AP-ACSF (4-aminopyridine artificial cerebrospinal fluid) into a 500 mL Erlenmeyer flask.
- 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.
- Start bubbling the 4AP-ACSF in the Erlenmeyer flask and in the syringe with a 95% O2/5% CO2 gas mixture.
- Let the 4AP-ACSF flow through the perfusion tubing into a beaker until there is no air inside; then stop the solution flow.
- 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's inlet and outer reservoir.
- 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.
- 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.
- 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.
- Once 4AP-ACSF is flowing through the cannula, turn on the thermostat. Set the heating cannula to 37 °C and the MEA base to 32 °C to achieve a 32 - 34 °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 °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 °C to avoid overheating the brain slice.
- 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.
2. MEA Live Mapping
- 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 (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'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.
- Take a picture of the brain slice using a camera mounted on an inverted microscope stage.
- 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.
- 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.
- 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.
- 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.
- Press the Save button: the software generates a result folder named #EXP_LabelledElectrodes containing a table reporting the selected electrodes and ROIs.
3. Recording and Electrical Modulation of the Epileptiform Activity
- Allow the brain slice to stabilize within the recording chamber for 5 - 10 min before recording.
- 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.
- 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.
- 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.
- Determine the stimulus intensity.
- In the stimulus control software, use the main panel to design a square biphasic positive-negative current pulse duration of 100 µs/phase.
NOTE: CAUTION! Direct current stimulation requires a balanced charge pulse to avoid damaging the equipment.
- 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 µA/phase and increase by 50 - 100 µA steps at each trial until stimulation can reliably evoke interictal-like events in the parahippocampal cortices (check the signals visualized by the recording software).
- Electrical modulation of limbic ictogenesis
- 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 ≤20 %.
- After stimulation stops, verify network recovery to pre-stimulus condition by recording at least 4 ictal discharges (as in step 3.4).
