Microelectrode Array-Based Assessment of Neuronal Networks in Mouse Spinal Cord Slices

Take a microelectrode array, or MEA, well, containing aCSF. Position a mouse spinal cord slice in the well and secure it with a weighted net.

Place the MEA well in a recording system. Under a microscope, ensure maximal contact between the MEA electrodes and the superficial dorsal horn or SDH, a region rich in interneurons.

Maintain a continuous aCSF flow to equilibrate the tissue.

Neurons communicate through action potentials. Sodium ion influx depolarizes the membrane, followed by potassium ion outflow that causes repolarization. The membrane briefly hyperpolarizes, preventing a new action potential until the resting potential is restored.

Record spontaneous neuronal activity from the MEA electrodes. Signals co-occurring across multiple electrodes indicate a network of synaptically linked neurons.

Introduce a potassium channel inhibitor to prolong depolarization that leads to increased action potential frequency and synchronous rhythmic activity across the network.

More electrodes showing coincident signals indicate the inhibitor-mediated synchronous activity.

To begin recording dorsal horn activity, transfer the slice from the incubator to the MEA well using a large-tip tip Pasteur pipette filled with artificial CSF, and add additional artificial CSF.

Use a fine, short-hair paintbrush to position the slice over the 60-electrode recording array. Then, place a weighted net over the tissue to hold it in place and promote good contact with MEA electrodes.

Place the MEA in the recording head stage. Check the position of the tissue over the electrodes using an inverted microscope to confirm that as many electrodes as possible are under the superficial DH. Ensure that at least two to six electrodes do not contact the slice.

After connecting the camera to the device, take a reference image of the slice relative to the MEA for use during the analysis. Then, press Start DAQ in the recording software and confirm that all electrodes receive a clear signal.

Next, attach the perfusion inlet and outlet lines to the MEA well filled with artificial CSF, and turn the perfusion system on. Check the flow rate and ensure that the outflow is sufficient to prevent overflow of the superfusate. After equilibrating the tissue for five minutes, record the raw baseline data for five minutes.

Move the perfusion inlet line from artificial CSF to a 4-aminopyridine solution, and wait for 12 minutes for the 4-aminopyridine-induced rhythmic activity to reach steady state. Then, record five minutes of 4-aminopyridine-induced activity, and be prepared for the subsequent recordings to test the drugs or check the stability of 4-aminopyridine.