Recording Electrophysiological Activity of the Neuronal Network in a Neuron-Astrocyte Co-culture

Begin with a multi-electrode array, or MEA, plate. Each well contains a centrally positioned array of electrodes.

The array is coated with a culture substrate to facilitate cell adhesion.

Add a suspension of motor neurons and astrocytes as a droplet over the array.

Incubate to allow cell attachment, then add a warm co-culture medium supplemented with a small molecule that maintains cell viability.

During incubation, astrocytes secrete factors that promote neuronal maturation and synapse formation, resulting in a neuronal network.

Place the MEA in a recording device under physiological conditions to monitor neuronal activity.

Neurons transmit electrical signals, called action potentials, that travel along their axons.

Upon reaching the synapse, the signal triggers neurotransmitter release, propagating the signal to the next neuron.

Electrodes within each well extracellularly record the signals generated by synaptically connected neurons.

The detection of synchronized rhythmic electrical signals confirms the establishment of a neuronal network.

On the day of plating or before, start coating the 24-well MEA plates by first diluting PLO in water or PBS. Then, add 15 to 20 microliters of PLO to each well, forming a droplet on the center of the well, covering the electrode area, ensuring not to damage electrodes with the pipette tip.

Add water to the compartments' surrounding wells, ensuring sufficient humidity, and incubate PLO at 37 degrees Celsius for 1 to 2 hours. Then, using a plastic micropipette tip, aspirate as much PLO as possible without touching the electrodes. Next, wash the well with 250 microliters of water, three times.

Using a pipette tip, remove as much water as possible and let the surface dry with the lid removed. Next, add 15 to 20 microliters of diluted laminin to cover each electrode array. After adding water to the humidity compartments and replacing the lid, incubate at 37 degrees Celsius for a minimum of two hours up to overnight.

On the day of plating, after rinsing the motor neurons and astrocyte cultures once with PBS, add 0.05% trypsin and incubate at 37 degrees Celsius for about 5 minutes to lift cells. Collect cells into a 15-milliliter conical tube, containing trypsin inhibitor, and wash plates with medium or base to ensure that all the cells are collected.

Pellet down the cells by centrifugation, and then using a 1,000-microliter pipette, resuspend motor neurons and astrocytes with a co-culture medium containing 20 micromolar of ROCK inhibitor to generate 1 milliliter of a single-cell suspension. Using a hemocytometer, count the motor neurons and astrocytes, and while counting, cap the cell suspensions and place them in a styrofoam rack at 4 degrees Celsius.

Centrifuge 1 milliliter of both cell suspensions at 300 times g for 5 minutes. Calculate the desired volume, and re-suspend the pellets. Combine the required volume of neuron and astrocyte suspensions in equal ratios, and mix gently by pipetting twice to combine thoroughly.

Then, remove laminin from each well of the plate and transfer 10 microliters of the final combined cell suspension to each well, forming a small droplet covering the electrode array. Incubate the plates with an undisturbed cell droplet at 37 degrees Celsius for 20 to 30 minutes to form initial attachments on the plates.

Then, pipette 250 microliters of warm co-culture media containing ROCK inhibitor down on the wall of each well, and pipette the same volume on the opposite wall of each well, and incubate the plate at 37 degrees Celsius for 24 to 36 hours. The next day, examine the plates for debris or dead cells, and if required, exchange the medium with a fresh co-culture medium containing ROCK inhibitor.

Otherwise, perform half-medium exchanges twice a week using a co-culture medium without a ROCK inhibitor, starting on co-culture day 2. To perform recording, start as soon as possible after co-culture day 1 by setting the temperature to 37 degrees Celsius and carbon dioxide at 5%. Then, transfer plates to the recording machine, and equilibrate for at least five minutes before recording. Record baseline activity every other day or weekly over 1 to 15 minutes, depending on the experimental design.