The overall goal of this procedure is to demonstrate how to culture and record electrophysiological activity of auditory neurons on multi-electrode arrays. This method can help answer key questions in the hearing research field, such as characterizing the electrophysiological property of auditory neurons and the stimulation by the electrode areas. The main advantage of this technique is that it allows for extracellular non-invasive simultaneous recording of the neuronal activity of large number of auditory neurons.
The implications of this technique extend towards therapy. In fact, they could be used to implement the electroneuron interface of your prosthetics like cochlear implant to restore hearing in deaf patients. To begin this procedure, prepare the ECM coating solution by first thawing the ECM mix on ice.
Then dilute the ECM mix in basic culture medium at a ratio of one to 10, and store it on ice. For new MEAs in a laminar flow hood, immerse them in 70%ethanol for 30 seconds. Subsequently, wash them with distilled water for 30 seconds.
Then let the MEAs dry for 30 minutes. To coat the MEAs, pipette 50 microliters of coating solution over the MEAs with a cold 200 microliter pipette tip. Then allow the MEAs to go for 30 minutes to one hour at room temperature.
After that, remove the coating solution. Add 100 microliters of culture medium supplemented with 10%FBS and five nanograms per milliliter BDNF and leave it at room temperature until plating the tissue. Next, place the Petri dish containing the coated MEAs in a large Petri dish and add a small Petri dish containing PBS for humidification.
In this procedure, sterilize the pup's head with 70%ethanol. Then cut the connection between the skin and the skull along the sagittal line. Next, cut the skull sagitally and remove the brain.
After that, cut the temporal bones from the skull and place them in a Petri dish containing sterile ice-cold HBSS. Under a dissection microscope, dissect the tympanic bulla using a pair of fine forceps and isolate the inner ear. Then, remove the bone of the cochlea.
Next, remove the spiral ligament and SV together by holding the basal portion of the spiral ganglion and the SV with forceps and slowly unwinding the SV from base to apex. Separate the organ of Corti from the spiral ganglion in modiolus by holding the basal portion of the spiral ganglion and the organ of Corti with forceps and slowly unwinding the organ of Corti from base to apex. Then, cut the lateral explants from the spiral ganglion using forceps or fine micro-dissection scissors or knives.
Now transfer the spiral ganglion explants and the organ of Corti onto the MEA. Then, place SG explants over the electrode area and the organ of Corti approximately five millimeters away from the electrode area. Place the explants and the organ of Corti onto the MEAs while avoiding damage to the tissue.
Afterward, place the MEAs carefully in the incubator and culture at 37 degrees Celsius and 5%CO2. The next day, visually inspect the explants for their attachment to the MEAs. Then, add 100 microliters of culture medium containing 10%FBS and BDNF daily for five consecutive days.
On day six, add two milliliters of culture medium containing 10%FBS and five nanogram per milliliter BDNF and culture the tissue for an additional 13 days. After a collective 18 days of culture, wash the MEA culture with the extracellular solution prepared earlier at room temperature. Then, dry the contacts of the MEA chip with a piece of tissue and mount the MEAs on the MEA holder.
Finally, install the MEA on the recording setup. Afterward, add 300 microliters of the extracellular solution and wait for 10 minutes to allow the system to stabilize before recording. Now, record the spontaneous activity for two minutes from all electrodes and identify the active electrodes.
Then, identify the electrodes responding to stimulation. To exclude the stimulation artifact, stimulate from the same electrode 10 times. If the culture responds at least eight out of 10 times, it can be assumed as a positive response upon electrode-induced stimulation.
To identify a background noise, apply TTX to the culture at a concentration of one micromolar to block the voltage gated sodium channels and then record for two minutes. Here are the traces of the original recordings of six out of 63 electrodes which show spontaneous activity and this is the raster plot of the six electrodes after spike detection. Each bar represents one action potential.
This raster plot includes all of the active electrodes. The activities are recorded from 63 electrodes for two minutes. This figure illustrates that a biphasic stimulus with a total duration of 80 microseconds and an amplitude of 80 micro amps was used for culture stimulation from one electrode.
This representative example of raw data traces shows the action potentials, all without responses after stimulation. And here's the spiral ganglion culture on the MEAs immuno-stained for the neuronal marker, TUJ, at the end of the experiment to visualize the neuronal coverage of the electrode area. The electrode used for the stimulation is indicated in green and the responding electrodes are indicated in red.
Once mastered, this technique can be done in 50 minutes if performed properly. Though these methods can provide insight into spiral ganglion neuron electrophysiology, it can also be applied to other systems such as brain or spinal cord cultures. After development, this technique paves the way for researchers in the field of material science and nanotechnology, quick blot surface modification of electrodes of neuronal recording and stimulation.
After watching this video, you should have a good understanding of how to culture and record electrophysiological activity, by arranging a neuron explant on multielectrode arrays.
