Analyzing Neural Activity in Primary Murine Spiral Ganglion Neurons Using Multielectrode Arrays

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Electrophysiological Recordings to Investigate Spontaneous and Electrode Stimulation Dependent Activity

1. Wash the multielectrode array (MEA) culture with extracellular solution, at room temperature (RT).
2. Dry the contacts with a tissue and mount the MEA on the MEA setup.
   NOTE: To keep the culture humid during the mounting, add a small drop of extracellular solution to the culture.
3. Add 300 µl of extracellular solution and wait for 10 min before recording, to allow the system to stabilize.
4. Record spontaneous activity for 2 min from all electrodes by pressing on the record/acquire button of the software and identify recording electrodes.
5. MEA electrode stimulation: Select the amplitude/duration/shape of the stimulus on the appropriate software and apply it to several electrodes consecutively. Select the electrodes based on the ones showing spontaneous activity (as in step 1.4). Record from all the remaining electrodes.
6. To exclude stimulation artifact, stimulate from the same electrode 10 times. If the culture responds at least 8 out of 10 times, it can be assumed as a positive response upon electrode induced stimulation.
7. To identify background noise, apply Tetrodotoxin (TTX) to the culture at a concentration of 1 µM, to block voltage-gated sodium channels and record for 2 min. Use this to perform spike detection (2.1 and 2.2).

2. Data analysis

1. Using the appropriate software detect spontaneous activity for each electrode employing a detector based on standard deviations and a subsequent discriminator. Activity appears as fast voltage transients (<5 msec).
2. Choose a threshold that results in no activity when analyzing the TTX treated samples. Adapt the threshold value for each experiment to discriminate between false positive and false negative detections.
3. Using appropriate software observe the detected neuronal activity of each electrode as a raster plot according to standard procedures (see Figure 1A–C).
4. Determine and display the total network activity by summing up all detected events within a sliding window of 10 msec, shifted by 1 msec steps.
5. Detect stimulation-induced activity by displaying the raw data using appropriate analysis software. Identify the single spikes offline manually. Single spikes appear as fast voltage transients, occurring after the stimulation artifact (arrowhead in Figure 1E). An example is shown in Figure 1E.
6. Depending on the experiment, analyze the number of responding electrodes, the threshold needed to achieve a response, number of action potential per electrodes and other parameters of interest.