Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Patients must give informed written consent prior to operation, and necessary ethical agreements must be in place prior to the experiment. Concerning the representative results, all studies involving human participants were reviewed and approved by Charité-Universitätsmedizin, Berlin (EA2/111/14).

1. Preparation of 10x solutions

NOTE: Due to difficulties in planning access to human brain tissue, it is recommended to prepare 10x solutions as described here. Alternatively, final 1x solutions can be prepared freshly by adding individual substances in final concentration to double-distilled water (ddH₂O).

1. For individual 10x solutions, add substances to ddH₂O according to Table 1 and stir until dissolved.
2. Use 10x solutions up to 1 month after preparation (up to 1 year for frozen 10x choline aCSF).
3. For 10x choline aCSF, prepare 50 mL aliquots of 10x 1.1 choline aCSF (Table 1) and freeze at -20 °C or -80 °C until further use.
   NOTE: Do not add glucose and CaCl₂ to 10x 1.1 choline aCSF to prevent contamination with bacteria and precipitation of calcium carbonate.
4. 10x solution 2 can be used for all 1x final solutions, whereas 10x solutions 1.1–1.4 are customized and named accordingly (Table 1).

2. Preparation of 1x final solutions

NOTE: Final 1x solutions should be prepared fresh or earliest as possible on the day before use. All final solutions should be carbogenated with 5% CO₂ and 95% O₂ using a glass gas disperser to enrich solutions with oxygen, and adjust the pH to 7.4 (max = 7.4 ± 0.2).

1. Choline aCSF for transport and preparation
   1. For the final 500 mL solution, thaw a 50 mL aliquot of the 10x solution 1.1 aliquot for choline aCSF in 37 °C water bath.
   2. Add the thawed 50 mL aliquot of the 10x solution 1.1 and 50 mL of 10x solution 2 to approximately 300 mL of ddH₂O.
   3. Add final concentrations of glucose and CaCl_2, then stir until dissolved (Table 1, solution 1.1).
   4. Add ddH₂O to a final volume of 500 mL and measure osmolarity (300 mOsm ± 10 mOsm).
   5. Optionally, use a filter to sterilize the solution (see discussion on prolonged slice viability under sterile conditions).
   6. Fill a separate bottle with approximately 100 mL of 1x choline aCSF for transport from the operation room to laboratory.
   7. Optional: depending on the transport time from the operation room to laboratory, consider using gas-tight bottle caps to ensure stable pH of aCSF during longer transportation periods.
   8. Store the final solution at 4–8°C until further use.
   9. On the day of operation, chill 1x choline aCSF on ice and carbogenate for at least 10–15 min using a glass gas disperser connected to carbogen gas (5% CO₂, 95% O₂).
      NOTE: Consider keeping a gas bottle accessible to the operation room in the case of longer waiting times, which will require re-carbogenation of the transport solution. However, we have transported hippocampal tissue without re-carbogenation before long and short transportation times (15 min vs. 60 min) and did not observe differences in induction of epileptiform activity.
2. aCSF for storage and recording
   1. For a final 2 L solution, add 200 mL of 10x solution 1.2 (aCSF) and 200 mL of 10x solution 2 and glucose (Table 1) to ~1500 mL of ddH₂O.
      NOTE: Volumes of the final solutions depend on applied experiments and the type of chamber used for storing and recording.
   2. Add ddH₂O to a final volume of 2 L and measure osmolarity (300 mOsm ±10 mOsm).
   3. Prewarm the solution to 35 °C and carbogenate for at least 10–15 min before use.
3. HighK⁺+4-AP aCSF for induction of burst activity
   1. For a final 1 L solution, add 100 mL of 10x solution 1.3 (highK⁺+4-AP aCSF) and 100 mL of 10x solution 2 to ~700 mL of ddH₂O.
   2. Add glucose and 4-AP (final concentration = 100 µM) according to Table 1.
   3. Add ddH₂O to final volume of 1 L and measure osmolarity (300 mOsm ±10 mOsm).
   4. Prewarm the solution to 35 °C and carbogenate for at least 10–15 min before use.
4. LowMg²⁺+BIC aCSF for induction of seizure-like events (SLEs)
   1. For a final 1 L solution, add 100 mL of 10x solution 1.4 (lowMg²⁺+BIC aCSF) and 100 mL of 10x solution 2 to ~700 mL of ddH₂O.
   2. Add glucose and BIC (final concentration = 10 µM) according to Table 1.
   3. Add ddH₂O to final volume of 1 L and measure osmolarity (300 mOsm ± 10 mOsm).
   4. Prewarm the solution to 35 °C and carbogenate for at least 10–15 min before use.

3. Preparation of interface chamber

1. In an interface chamber, slices rest on three layers of filter paper to ensure sufficient amount of solution below the slice. To do this, cut two ~4 cm x ~2 cm pieces of filter paper for each slice-holding compartment (the described interface chamber consists of two compartments) and place them on top of each other.
2. Place thin cotton strings around the 4 cm x 2 cm filter papers inside the compartments to break the tension of the solution. Ensure even flow (here, black nylon tights cut to thin-cut strings ~10 cm in length are used; for placement, see Figure 1).
3. Place small pieces of filter paper on top of the larger filter papers inside the slice-holding compartments. Small filter tissue pieces should be roughly the size of one brain slice (~1.5 cm x ~1.0 cm) and will enable further handling of individual slices. Place three to four small filter paper pieces in each compartment.
4. Ensure an aCSF flow rate of 1.8 mL/min with a peristaltic pump.
5. Carbogenate and prewarm the interface chamber to ~35 °C (final temperature of the slice should be ~32 °C).

4. Set-up of preparation area

NOTE: Preparation can be performed under sterile conditions to avoid contamination and elongate slice survival. However, not all vibratomes fit under a sterile hood, and other measures are required to reduce contamination during preparation. This section describes some of these measures.

1. Wipe the preparation area with 70% EtOH and place either aluminum foil or sterile covers on top of the area.
2. Prepare super glue, two sharp tweezers, a spatula, a scalpel with blades, and a blade for rough cutting of the brain tissue. Tools can be sterilized prior to the procedure to reduce contamination.
3. Wipe the buffer tray and specimen plate of the vibratome with 70% EtOH. Once the buffer tray is fully dry, cover it with aluminum foil and place the tray in the ice bath. Fill the ice bath with crushed ice and keep at -20 °C until preparation.
4. Wipe the vibratome and razor blade with 70% EtOH and calibrate the vibratome to minimize vertical vibrations and tissue damage during slicing procedure.

5. Tissue slicing and storing

1. Directly after resection, place the tissue immediately in cold, carbogenated choline aCSF and transport quickly to the laboratory.
   CAUTION: Wear gloves and a face mask at all times during preparation, since human brain tissue can contain potential pathogens. In addition, wearing a face mask when not working under a sterile hood will greatly reduce contamination of solutions and brain tissue.
2. Remove tissue from choline aCSF and cut away any burned portions of tissue.
3. Cut an even surface to glue tissue piece onto the specimen plate, while considering the cutting angle and tissue layers. Ideally, a hippocampal slice contains DG, CA1-4, and (if possible) subiculum.
4. Slice the brain tissue into 400 µm thick slices and adjust the amplitude and speed during cutting. Due to possible remaining pia mater, human brain tissue shows more resistance and may require slower cutting.
   NOTE: Slice thickness greatly affects either the available network (more neurons in thicker slices) or viability of the slice (penetration of solution into the slice). We have used 500 µm slices to increase the potentially available micro-network, and could not observe differences in induction of epileptiform activity. 300 µm slices are commonly used for patch-clamp experiments, though the induction of epileptiform activity in these slices have not yet been tested here. We use 400 µm as a standard slice thickness, though 300–500 µm slices may be sufficient.
5. Before collecting, use a scalpel to reduce the sizes of brain slices to fit into the recording chamber. For use of the membrane chamber (see section 6), slices should be maximally 1.5 cm x 1 cm. While reducing, consider the specific layers and connections needed to be intact for recording (e.g., for recording in the CA1 and DG, cut away the subiculum and surrounding white tissue).
6. Using a spatula and small forceps, carefully place slices in the interface chamber on small filter papers and let them rest for ~1 h in aCSF until recording.
7. Slices can be recorded for up to 20 h (even longer when under sterile conditions).

6. Recording of epileptiform activity

1. In the membrane chamber (submerged type recording chamber), place the brain slice on a transparent semipermeable membrane, which is glued to a plastic ring²⁴. For this, use super glue to attach the plastic ring to the membrane of a cell culture insert.
2. Use a scalpel to remove any membrane on the outside of the plastic ring. Ensure that the membrane is even and fully attached to the ring before placing the membrane in the chamber.
   NOTE: The membrane can be stored in ddH₂O at 4–8 °C and reused for up to 1 month. Keep the membrane wet at all times.
3. Both the inflow and outflow of the membrane chamber are connected to tubes for solution supply. Place the tubes in a peristaltic pump so that the inflow and outflow move in opposite directions.
4. Place the inflow and outflow tube in carbogenated, prewarmed aCSF until all tubes and the chamber are filled with solution. Adjust the speed of the peristaltic pump to achieve an even flow rate of 10–13 mL/min.
   NOTE: The membrane chamber used here is a high flow rate, submerged type recording chamber enabling a solution flow of up to 14 mL/min²⁴. In the case of using a different submerged type recording chamber, flow rates need to be adjusted. However, for induction of epileptiform activity, it is highly recommended to use the membrane chamber.
5. Use a heating element connected to the inflow in close proximity to the membrane chamber to ensure a stable temperature of 32 °C.
6. Prepare 1–2 MΩ glass pipettes using a vertical puller. Fill pipettes with 154 mM NaCl solution and place them in an electrode holder.
7. Using tweezers and a spatula, remove a hippocampal slice from the interface chamber by taking the slice with the small filter paper and placing both in a Petri dish filled with carbogenated aCSF. Remove the small filter paper from the hippocampal slice, and (if necessary) apply some force using a pipette to separate the slice from the filter paper. Be careful not to flip the slice.
8. Place the slice in the recording chamber and hold it in place using slice mesh.
   NOTE: Due to Bernoulli’s principle, in the used submerged type membrane chamber, slices are usually stable without use of an additional slice mesh.
9. Place electrodes in the region and layer of interest (here, CA1) and begin recording.
10. Record field potential activity in current clamp mode with a sampling rate of 10–20 kHz and low-pass filtered at 2 kHz.
11. Record basal activity in aCSF for up to 5 min.
12. Switch the inflow tubes from aCSF to highK⁺+4AP or lowMg²⁺+BIC aCSF and the outflow tube to a waste container to prevent mixing of solutions. After 2 min, place the outflow tube in the same solution as the inflow to conserve solution.
13. Burst activity induced by highK⁺+4-AP should be visible 2–5 min after the wash in. However, induction of SLEs by lowMg²⁺+BIC can take up to 30 min. If necessary, change the positions of electrodes carefully to obtain optimal results.
14. Once in the final position, record baseline activity for at least 20 min. If you record SLEs, consider longer baseline recordings due to low frequency of SLEs.
15.  In the case that baseline activity is stable (plateau of event frequency), wash in the desired drug. Note that due to the high flow rate wash in of drugs takes only 2–5 min, allowing for fast solution exchange.
16. Record activity during  drug application of at least 20 min, following wash out. Activity should be stable for at least 60–90 min, allowing for longer recordings.

7. Analysis

1. Analysis of frequency and amplitude can be performed with any available software. So far, we have not been able to establish a reliable automatic analysis of SLEs or burst activity, and instead used a semi-automated analysis with visual confirmation of identified activity.
2. Burst activity is characterized by biphasic, positive, and negative deflection and a duration of ≥100 ms. All events visually identified as burst activity (e.g., semi-automatically by threshold analysis) should be manually indicated for further analysis of event frequency (inter-event interval, IEI), amplitude, and total number of events during the analyzed time frame.
   NOTE: Due to the high frequency of burst activity, the last 5 min of each application phase is typically analyzed²⁰.
3. SLEs can be analyzed as described in Heuzeroth et al. Identified SLEs can be further analyzed for duration, amplitude, spike frequency, and duration of tonic (high frequency spiking) vs. clonic (low frequency spiking) phase duration. SLEs that are <10 s in duration should be excluded from analysis.
   NOTE: For preclinical evaluation of possible antiepileptic substances, effects on burst activity (induced by highK⁺+4AP) are being investigated, due to established induction in resected hippocampal tissue. Preliminary results on induction of SLEs using lowMg²⁺+BIC have been reported (Figure 2), though analysis of this data is not included here.