All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Mounting Spinal Cord Tissue Slices on MEAs
- Warm up sterile nutrient medium in the incubator (37 °C, lightly unscrewed lid for oxygenation).
- Position the multi-electrode array or MEA with sterile tweezers with rubber-covered tips at room temperature (RT) in the petri dish under a stereo microscope with the electrode array in focus. Center a 6 µl droplet of chicken plasma on the clean, dust-free, and sterile electrode array. Using a small spatula, carefully slide two spinal cord sections with ventral sides facing each other into the plasma droplet. Do not touch the electrodes with the spatula.
- Add 8 µl of thrombin around the chicken plasma droplet. Using the thrombin pipette tip, carefully mix and spread the chicken plasma/thrombin mixture around the two slices. Again, do not touch the brittle electrode array directly. Just before coagulation, aspirate excess chicken plasma/thrombin.
- Cap the petri dish to retain high humidity while the MEA/culture assembly sits for about 1 hr in a humidified chamber inside the incubator at 37 °C.
- Carefully add 10 µl of nutrient medium to the culture chamber, cap the petri dish, and put back into the incubator for about 30 min.
- Place each MEA/culture assembly with sterile, rubber-covered tips into a roller tube, add 3 ml of nutrient medium, and tightly close the lid. Place the roller tube in the roller drum, rotating at 1 - 2 rpm in the incubator in a 5% CO2-containing atmosphere at 37 °C.
- Change half of the nutrient medium after 7 days in vitro (DIV) and afterward 1 - 2 times per week.
2. Mechanical Lesions
- Take the MEA/culture assembly with sterile, rubber-covered tips out of the roller tube and place in a petri dish without nutrient medium under a stereo microscope with the tissue in focus. Visually verify that the two slices are fused.
- Hold the MEA/culture assembly steady by placing tweezers with rubber-covered tips on the MEA.
- Place a scalpel blade in the groove of the MEA close to the tissue slices. Hold the scalpel rather horizontally.
- Lift the scalpel handle up but let the scalpel blade stay in the groove of the MEA in such a way that the blade "rolls" from base to tip and thereby cuts through the tissue covering the groove.
- Severe any residual tissue connections with a 25 G needle tip if necessary. Work only in the area within the groove and do not touch the tender edges.
- Put the MEA/culture assembly back into the roller tube. Provide 3 ml of fresh nutrient medium to the cultures and place them back into the roller drum in the incubator.
3. Electrophysiological Recordings of Spontaneous Activity
- To investigate functional regeneration among the two spinal cord slices after the chosen number of DIV, mount the MEA/culture assembly in a recording chamber on a microscope and apply about 500 µl extracellular solution (see materials list).
- Wait 10 min before the first recording to allow the system to stabilize.
- Record basic spontaneous activity 2x for about 10 min from each activity-detecting electrode of the MEA at RT.
- To ensure stable, extracellular conditions, exchange extracellular solution after every recording session.
- To disinhibit the network, apply an extracellular solution containing strychnine (1 μM) and gabazine (10 μM). Wait for at least 2 min before starting the recording.
4. Data Analysis
NOTE: For the detection of the extracellularly recorded action potentials, use a detector based on standard deviations and a subsequent discriminator for each electrode.
- Display the detected neuronal activity of each electrode in a raster plot according to standard procedures (Figure 1F).
- Determine and display the total network activity for each slice by summarizing the number of events in the appropriate channels within a sliding window of 10 msec, shifted by 1 msec steps (Figure 1G).
- Detect in each slice individual bursts (clusters of activity that appear on several electrodes). Therefore, set a minimal peak activity threshold in the corresponding total network activity and define each burst start and burst end. For example, an appropriate threshold is 25% of the average burst amplitude, and a burst start can be defined as being the first event in a time window of at least 5 msec, and a burst end by being the last event in a time window of at least 25 msec.
- To quantify functional regeneration, calculate the percentage of synchronized bursts between the two slices. To do this, calculate the latency between a burst in one slice and the following burst in the other slice.
NOTE: A burst pair is termed "synchronized" when the latency is smaller than the average burst length. Especially in co-cultures with a lot of activity, the possibility exists that both sides coincidentally initiate a burst in the chosen latency window. This fact has to be taken into account in the quantification of the percentage of synchronized bursts.
