Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

All methods described here have been approved by the Italian Ministry of Health (authorization n. 860/2015-PR) in compliance with the EU directive 2010/63/EC on animal welfare.

1. Preparation of the MEA/Custom Chamber Assembly

Note: Start 2 days before the experiment. Use a MEA without a ring (see Materials Table).

1. Clean the MEA (duration: 35 – 40 min).
   1. Dissolve an enzymatic cleaner powder into warm distilled water. Brush the warm cleaning solution onto the MEA surface then soak the MEA into the warm cleaning solution for at least 3 h.
   2. Rinse the MEA thoroughly with distilled water and dry it using a lint-free no-scratch tissue.
2. Assemble the MEA with the recording chamber (duration: 10 min + overnight).
   1. Evenly spread the elastomeric sealant on the bottom surface of the recording chamber. Make sure there are no bubbles.
   2. Mount the recording chamber onto the MEA with the help of tweezers and apply gentle pressure. If there are any bubbles, slightly move the chamber in a circle while applying gentle pressure with fingers until all the bubbles have disappeared. Spread a sealant layer all the way around the outer border of the recording chamber.
      Note: CRITICAL! Do not cover the MEA contacts with the sealant.
   3. Place the MEA/chamber assembly inside a 15 cm Petri dish. Place a 5 mL Petri dish or a 10 mL beaker filled with distilled water close to the MEA. Cover everything to maintain a moist environment. Let cure at room temperature overnight.
      NOTE: The protocol can be paused here.
3. Coat the MEA to make it hydrophilic (duration: 5 – 10 min + overnight).
   1. Place the MEA into a 15 cm Petri dish. Pour 50 µL of poly-D-lysine onto the MEA recording area. Close the Petri dish, seal it with sealing film, and store overnight at 4 °C.
   2. Rinse the MEA thoroughly using distilled water and store the MEA at 4 °C in distilled water.
      NOTE: The protocol can be paused here. Coated MEAs may be stored and reused for several experiments. Routinely cleaning and re-coating the MEA helps remove tissue debris and improve signal-to-noise ratio.

2. Preparation of Stock Solutions

Note: Start 1 day before the experiment (duration: ~2 h). Stock solutions help speed-up the experiment on the experimental day. They can be prepared in advance and stored. See Table 1 for concentration factor and composition. Refer to Table 2 for composition of final solutions.

1. Dissolve the chemicals in distilled water at room temperature. Filter the stock solutions with a filter bottle (filter diameter: 0.22 µm).
2. Store at 4 °C (see Table 1 for recommended maximum storage time).
   NOTE: The protocol can be paused here.

3. Preparation of Agar

Note: Start 1 day before the experiment (duration: ~1 h).

1. Pour 250 mL of distilled water into a 500 mL beaker and start stirring at 350 rpm. Add 5 g of agar and wait until it is completely dissolved.
2. Heat the solution at 250 – 300 °C. Let the water evaporate until the solution is ~200 mL to yield a 2.5% w/v agar solution.
   NOTE: The temperature should be high enough to let water evaporate without bubbling.
3. Pour the agar solution onto a 200 mL plastic square box of ~1.5 cm-high walls and let it cool down at room temperature until it becomes solid.
4. Cover the box and store it at 4 °C. The agar block is good for several weeks.
   NOTE: The protocol can be paused here.

4. Preparation of the Frozen Plane

Note: Start 1 day before the experiment.

1. Fill a flat-bottom stainless steel bowl with distilled water up to 0.5 – 1 cm below the rim.
2. Store at -20 °C overnight.

5. Preparation of Neuroprotective Cutting ACSF

Note: Start 1 day before the experiment or the same day of the experiment (see Table 1 and Table 2) (duration: 30 min).

1. Pour 200 mL of distilled water in a 500 mL beaker and start stirring using a magnetic stirrer at 350 rpm.
2. Add 50 mL of stock B and 50 mL of stock C (see Table 1).
3. Add 3 mM pyruvic acid, 208 mM sucrose, 10 mM D-glucose, and 1 mM L-ascorbic acid.
   NOTE: The actual volume of pyruvic acid depends on the density and purity and may vary with the batch. Always calculate the required volume when opening a new batch.
4. Cover with sealing film and let stir for 30 min.
5. Transfer the solution to a 500 mL volumetric flask and add distilled water to fill the flask.
6. Seal the volumetric flask with sealing film and gently invert 3 – 5 times until the solution is evenly clear.
7. Transfer the cutting ACSF into a glass bottle with cap.
8. Store the cutting ACSF at -80 °C for 20 – 30 min or at 4 °C overnight to cool down to 4 °C.
   NOTE: The protocol can be paused here if overnight cooling is preferred. Otherwise the time required for cooling the cutting ACSF may be used to pursue step 6.

6. Preparation of the Holding ACSF

Note: Prepare the solution fresh on the day of the experiment (duration: 5 – 10 min). The holding ACSF will be used to rinse the brain slices from the cutting ACSF during the slicing procedure (see step 8.16) and for brain slice long-term storage and pre-warming. To rinse the brain slices, 100 mL of holding ACSF is sufficient. The holding and pre-warming chambers used in this protocol are custom-made and contain a volume of 300 mL and 100 mL, respectively (cf. Figure 1A). With this setting, 500 mL of holding ACSF is sufficient. Chambers obtained from commercial sources may contain a different volume, in which case the overall amount of holding ACSF to be prepared should be adjusted accordingly.

1. Pour 200 mL of distilled water in a 500 mL volumetric flask.
2. Add 50 mL of stock A, 50 mL of stock B, 6.5 mL of stock M, and 1 mM L-ascorbic acid. Gently agitate using rotating movements until L-ascorbic acid is completely dissolved.
3. Add distilled water to reach the final volume, seal the volumetric flask with sealing film, and gently invert 3 – 5 times until the solution is evenly clear. Refer to Table 2 for the holding ACSF composition.

7. Preparation of the Experimental Benches

Note: This requires 15 min, plus 30 min to obtain a steady temperature of the warm bath.

1. Recording bench
   1. Start the warm bath, set it to 32 °C, and cover it to maintain its temperature.
      NOTE: The warm bath used in this protocol can be custom-made using a hard plastic box and an aquarium thermostat pre-set at the desired temperature. Steady desired temperature may be reached in ~30 min starting from room temperature. Since the pre-warming and incubating chambers sit at the bottom of the plastic box, the water level should be just enough to cover the thermostat so as to keep the chambers from floating.
   2. Assemble the holding and the pre-warming chambers (cf. Figure 1A), and pour the holding ACSF into them. Keep the holding chamber at room temperature and place the pre-warming chamber inside the warm bath. Start bubbling the solutions with 95% O₂/5% CO₂ gas mixture and remove any trapped bubbles using an inverted Pasteur pipette. Keep covered.
2. Slicing bench and vibratome
   1. Glue a small agar block onto the center of the specimen disc using cyanoacrylate glue.
   2. Put a 250 mL beaker into an ice bucket and pour cutting ACSF into it.
   3. Take two 100 mL beakers and pour 50 mL of holding ACSF into each. Leave the beakers at room temperature. These are the rinsing beakers.
   4. Start bubbling the solutions with a 95% O₂/5% CO₂ gas mixture.
   5. Mount the buffer tray onto the vibratome and surround it with crushed ice. Pour some ethanol on the crushed ice to aid in maintaining cold temperatures during the slicing procedure. After pouring ethanol, add more ice as needed.
      NOTE: A temperature of 2 °C is optimal.
   6. Place the bubbler inside the buffer tray, then assemble and mount the vibratome blade block.
      NOTE: Make sure that the blade is mounted at clearance angle of ~10°.
   7. Put crushed ice in a 500 mL beaker up to 2/3 of the volume and fill with distilled water.
   8. Take the frozen bowl out of the freezer, place it upside-down on the slicing bench, and cover it with a paper towel and a filter paper disc.
3. Anesthesia bench
   1. Place a small bowl in an ice-filled tray and pour cutting ACSF in it. Start bubbling with a 95% O₂/5% CO₂ gas mixture.

8. Brain Slice Preparation and Maintenance

Note: This protocol makes use of adult (4 – 6 weeks-old) male CD1 mice, but other strains (e.g., c57/bl6^17,18) can be used. We also later compare the experimental output obtained using mouse brain slices with that yielded by brain slices obtained from male Sprague-Dawley rats of the same age. The following steps refer to the preparation of partially disconnected brain slices in which CA3-driven fast interictal-like activity is restrained to the hippocampus proper and cannot propagate to the parahippocampal cortices, as described in²³. Hippocampus-cortex disconnection is a pre-requisite to pursue electrical modulation studies in using the hippocampus-EC brain slice preparation. The vibratome used refers to the model listed in the Materials Table. Other models may require a different procedure.

1. Anesthetize the rodent using isoflurane 5% in a carbogen gas mixture delivered to an anesthesia induction chamber at 2 L/min.
2. Under deep anesthesia (no reflexes in response to foot and paws pinch), extract the brain within 1 min following standard procedures as in²³. Place the brain into the small bowl containing equilibrated ice-cold cutting ACSF and let chill for 90 – 120 s.
   NOTE: Do not let the brain chill for too long, otherwise it may freeze.
3. Pour the ice-cold cutting ACSF into the buffer tray. Spread cutting ACSF onto the filter paper placed on the frozen bowl. Place the brain onto the filter paper and isolate the required brain tissue block by removing the cerebellum and cutting the frontal pole out straight.
4. With the help of a bent spatula, glue the dorsal side of the brain onto the specimen disc with the cut frontal side facing the agar block and the occipital pole facing the vibratome blade. Use a thin layer of cyanoacrylate glue. Place the specimen disc in the buffer tray immediately and secure it.
5. Adjust the sectioning range so as to move the vibratome blade all the way across the tissue up to the agar block. Adjust the specimen disc height to reach the blade level with the brain tissue block.
6. Discard the tissue sections until the hippocampus is clearly visible (usually ~ 900 µm). Then, set a slice thickness of 400 µm and start slicing to retain the tissue sections. For each brain section split the two hemispheres and trim the unnecessary tissue to obtain two brain slices. As the buffer tray is heightened during subsequent sections, fill the buffer tray lodgment with iced distilled water (see step 7.2.7).
7. Use an inverted Pasteur pipette to gently transfer the brain slices in the first rinsing beaker, empty the Pasteur pipette, and transfer the brain slices to the second rinsing beaker. Then, transfer the brain slices into the holding chamber and let them recover for at least 60 min.
   NOTE: Usually it is possible to obtain 6 – 8 brain slices overall. The protocol may be paused here.

9. Preparation of ACSF Containing 4-AP (4AP-ACSF)

Note: This requires 10 min for preparation, plus 20 min of warming. CAUTION! 4-AP is a convulsant drug and it is toxic. Handle with gloves and avoid spilling.

1. Pour 200 mL of distilled water in a 500 mL volumetric flask.
2. Add 50 mL of stock A, 50 mL of stock B, 5 mL of stock M, and 1 mM L-ascorbic acid. Gently agitate using rotating movements until the L-ascorbic acid is completely dissolved.
3. Add 500 µL of 4AP stock.
4. Add distilled water to reach the final volume, seal the volumetric flask with sealing film, and gently invert it 3 – 5 times until the solution is evenly clear.
5. Assemble the 4AP incubating chamber (cf. Figure 1A) and pour ~80 mL of 4AP-ACSF into it. Cover the chamber, transfer it to the warm bath, and start bubbling with a 95% O₂/5% CO₂ gas mixture. Remove any trapped bubbles using an inverted Pasteur pipette. Keep covered.
6. Wait until the 4AP-ACSF temperature is 30 – 32 °C (~20 min).
   NOTE: The protocol may be paused here if brain slices are still recovering.

10. Pre-warming and Incubation of Slices in 4AP (Duration: 90 min)

1. Check that the pre-warming ACSF and the 4AP-ACSF temperature is 30 – 32 °C.
2. Use an inverted glass Pasteur pipette to transfer 1 brain slice into the pre-warming chamber and let the tissue rest for 25 – 30 min. Then transfer the brain slice into the 4AP-ACSF incubating chamber and let it rest for 60 min.

11. Preparation of the MEA Set-up

Note: Start 15 min before recording.

1. Transfer the remaining volume of 4AP-ACSF into a 500 mL Erlenmeyer flask.
2. Place the Erlenmeyer flask on a shelf above the MEA 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 are sufficient.
3. Start bubbling the 4AP-ACSF in the Erlenmeyer flask and in the syringe with a 95% O₂/5% CO₂ gas mixture.
4. Let the 4AP-ACSF flow through the perfusion tubing into a beaker until there is no air inside; then stop the solution flow.
5. 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. Use a plastic Pasteur pipette to transfer 4AP-ACSF to the inlet and outer reservoir of the recording chamber.
6. 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 to minimize noise due to the metallic material of the cannula. When the inlet reservoir is adequately filled with 4AP-ACSF, drops falling from the perfusion system should not be visible.
7. Place the suction needle inside the reservoir and verify that there is negative pressure by submerging the suction needle into the ACSF; check for a constant low-frequency suction noise.
   Note: The suction needle should be placed so as to allow the 4AP-ACSF to flow just above the brain slice surface. A vacuum line or a low-noise vacuum pump can be used.
8. 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.
9. 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 temperature of 32 – 34 °C 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 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 11.9 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.
10. Place the external reference electrode in the recording chamber inlet reservoir.
    NOTE: Although MEA chips are equipped with an internal reference electrode, this is covered by the custom recording chamber. Thus, an external reference electrode must be used. A saturated KCl pellet is the most practical, since it is ready to use without need for chlorination.

12. MEA Live Mapping

1. 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.
   1. 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 small 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.
2. Take a picture of the brain slice using a camera mounted on an inverted microscope stage.
3. 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.

Fig 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. Please click here to view a larger version of this figure.

4. Click the Navegar 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.
5. 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.
6. 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.
7. Press the Save button: the software generates a result folder named #EXP_LabelledElectrodes containing a table reporting the selected electrodes and ROIs.

13. Recording and Electrical Modulation of the Epileptiform Activity

1. Allow the brain slice to stabilize within the recording chamber for 5 – 10 min before recording.
2. 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 stimulator and MEA amplifier are correctly connected as indicated by a green LED in the main panel of the stimulus control software. Please refer to the manuals of the specific instruments and software for additional details, cf. Materials Table.
3. Set up the stimulation in 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.
4. 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.
5. Determine the stimulus intensity.
   1. 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.
   2. Run a fast input/output (I/O) test to identify the best stimulus intensity. Deliver the stimulation pulse designed at step 13.5.1 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).
6. Electrical modulation of limbic ictogenesis
7. 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%.
8. After stimulation stops, verify network recovery to pre-stimulus condition by recording at least 4 ictal discharges (as in step 13.4).