The overall goal of this protocol is to demonstrate how to combine the use of micro fluidics with micro electrode arrays to generate the platform suitable for studying neuronal communication and axonal signal propagation. Hello, I'm Paulo Aguiar and I'm the PI of the Neuro engineering and Computation Neuroscience Lab at INEB, used to code for biomedical engineering at University of Porto, Portugal. Understanding information transmission in neuro circuits remains one of the biggest challenges in neuroscience.
Today, I'm going to show you how we can combine individual micro actuator arrays with micro fluidics and software tools developed here at our lab, to detect and characterize propagating action potentials. We use this setup in a variety of studies, namely neural coding and decoding, and in communications between sensory neurons and other cell types. We believe that the visual demonstration of this protocol is crucial.
Since micro fluidic and micro electrode arrays assembly is that difficulty in managing, and difficult to reproduce just by reproducing protocols. For the market, cultary sales on this platform is not straightforward, as the conventional ABAs, and it requires special attention to the cell sitting stuff, and culture magnets. This setup allows us to study several communication related properties, such as propagation velocity and direction, in a well controlled environment.
Although the recording procedure is simple, data analysis requires knowledge and the use of the right computational tools. On the day before cell sitting, start by preparing the micro fluidic devices, by cleaning down with vinyl tape. Gently press the tape against the device to reach all areas of the micro fluidic.
Air plasma clean the MEA for three minutes to clean the surface and make it more hydrophilic. Inside a laminar flow hood, submerge the micro fluidics in 70 percent ethanol. Allow them to air dry.
Place each MEA in a petri dish, and sterilize by 15 minutes of UV light exposure. Then, go to MEA's central area with 500 microliters of PEI solution. Incubate for at least one hour.
Inside a laminar flow hood, aspirate the PEI coating solution from the MEA surface. Then, wash the MEAs four times with one milliliter of sterile water. With the help of a sterile microscope, placed inside the laminar flow hood.
Center the MEA chip, and add one milliliter of sterile water to the central area of the MEA. Next, place the micro fluidic device on top of the MEA surface, and carefully align the microgrooves micro active grid of the MEA. Aspirate the excessive water, and incubate overnight at 37 degrees, to allow complete attachment.
On the next day, load the wells with laminin, and incubate again. After collecting the embryo susseds, proceed to the prefrontal cortex dissection. Start by carefully exposing the brain, using a pair of forceps.
Then, gently remove the brain from it's cavity. Cover it with cold H-HBSS. When present, remove and discard the olfactory bulbs.
Finally, dissect the prefrontal cortex. Ensure the complete removal of the meninges. Repeat this procedure for both hemispheres.
For the number of brains required for your study. Inside a laminar flow hood, collect the cortex fragments previously dissected in a total of five milliliters of H-HBSS. Then, transfer these fragments to a sterile 15 milliliter chronical tube.
Pipette, and have 2.3 milliliters of freshly prepared trypsin solution to the tube containing the cortex fragments. Mix the suspension, and incubate at 37 degrees for 15 minutes. Agitate every five minutes.
Then, discard the supernatant containing the trypsin, add H-HBSS containing FBS, to inactivate the remaining trypsin. Gently agitate. Let the cortex fragments settle down, and discard the supernatant.
Wash two times with H-HBSS, to remove the FBS, and discard the supernatant again. Add supplemented neural basal medium, and mechanically dissociate tissues by slowly pipetting up and down. Discard the remaining tissue by filtering the salt suspension through a cell strainer.
Then, mix 20 microliters of cell suspension with 20 microliters of trypan blue. Transfer 10 microliters to a Neubauer chamber, and count the number of viable cells. Immediately before cell sitting, remove laminin from all wells of the micro EF.Then, add the small volume of supernatant medium to each well of the axonal compartment.
Slowly seed five microliters of cell suspension to the somal compartment. Incubate at 37 degrees for one hour to allow cell attachment to the substrate. After one hour, gently fill each well with warm supplemented medium.
Add small volumes to avoid cell detachment. Then, transfer the micro EF to an incubator. Here is shown a culture at five days in vitro.
After one week, cultures start exhibiting spontaneous activity. Before performing recordings, use a temperature controller to eat the pre amp fire base plate. Then, place the micro EF on the preamplifier, and close and latch the lid.
To record, open the multi channel experimental software. Set the sampling rate to 20 kilohertz. Drag the filter and recorded boxes, and connect them sequentially to record both raw and filtered data.
Start the data acquisition to visualize the signals, and quick record. The recorded data can be quickly explored using multi channel analyzer. Here, the signal propagation along the microgrooves is already clear.
To identify and characterize this propagating events, open your recording in micro spike hunter. Click the file info button to access the recording settings, and select the set of micro electrodes for analysis. Set the threshold for propagating event detection.
And quick read data. The software will list all the detected sequences. The user can then assess the propagation velocity as the information, between pairs of micro electrodes, and based on their inter distance and cross correlation.
The chemo graph tool allows the user to manually estimate the propagation velocity. Here is shown a propagating event. Detected along four micro electrodes within a microgroove.
These events can be represented in a Rasser plot. Here is the propagating activity along 11 microgrooves for the first two minutes of recording. The high and the low activity microgroove are colored in yellow and red, respectfully.
Notice synchronization of the activity along the 11 microgrooves. The synchronization is independent of the rate of activity. As can be seen in the variations of the instantaneous firing rate of the two microgrooves.
Microgrooves also function as axonal signal amplifiers, as shown in the box and whiskers plot. We have shown you how to combine micro fluidics with MEAs, and how to culture neurons on them. Also, how to record and extract meaningful data from those recordings.
We hope this demonstration to be useful for your research, why you may wanted to study the communication in neural circuits.
