The goal of this experiment is to study the self-organization of healthy brain activity into neuronal avalanches, the hallmark of critical state dynamics in the cortex. This is achieved by combining a coronal layered cortex slice together with a midbrain slice, which provides developmentally important dopaminergic inputs to the cortex. As a second step, the Cortex midbrain co-culture is grown on a multi electrode array, which allows for multi-site stimulating and recording of neuronal activities in the culture.
Next, we let the co-culture flatten, expand, and attach to the MEA surface during the first one to two weeks in a custom designed incubator, which cycles the culture through exposure to normal air and culture. Fluid results are obtained that show the spontaneous self-organization of spatiotemporal local field potential bursts into neuronal avalanches. Based on MEA recordings and offline avalanche analysis that identifies the power law in avalanche sizes.
The main advantage of this technique over existing methods, such as dis associated cultures, is its systems, neuroscience aspect. We can co-culture many brain regions together and these multi region cultures establish a brain organization that is correct at the large scales such as cortical layers and projection systems. Applications of this technique extend beyond just the study of neurono avalanches.
For example, we can study stimulus in response at the network level under precisely controlled in vitro conditions. Visual demonstration of this method is critical as proper application of the plasma thrombin mixture and positioning the tissue on the MEA are difficult to learn. These steps require proper timing, the right temperature gradient, and avoiding touching the delicate MEA surface directly.
To prepare a sterile sealable glass chamber for recordings first cut threaded glass cylinders with a Teflon plastic cap, approximately two millimeters from the bottom of the thread. These are required for secure and tight culture chamber closure. Then clean the glass rings by rinsing them with water three times, then boiling for five minutes in 200 proof ethyl alcohol.
Set these aside to dry a silicon solution is required to attach the glass rings to the MEA surface. Use a cell guard 180 4 silicon elastomer kit and mix 15 milliliters of parts A and B thoroughly. Then let's sit for 15 minutes to remove air bubbles.
Separate the solution into one milliliter Eloqua, and store at minus 20 degrees Celsius for future use. Now glue a glass ring to the MEA by taking up one milliliter of silicon at 23 degrees Celsius in a syringe with a small gauge needle and applying it to the unpolished cut surface of the glass ring. Then center the glass ring on the MEA and apply an additional layer of silicon around the outside of the ring for a stronger seal.
Let this cure for one to two hours at about 60 degrees Celsius on a hot plate. Next, sterilize the MEA chamber and chamber caps under lamina flow hood by rinsing three times in deionized water than three times with 70%alcohol for the last rinse. Let the chamber sit for 10 minutes in the alcohol.
Follow this by exposing the chamber and cap interior to UV light for 10 minutes. Autoclave at 120 degrees Celsius for 45 minutes. Once sterilized, allow these to dry.
Now coat the MEA surface inside the culture chamber with poly D lysine. New meas as used in this experiment are rather lipophilic, hence coat by repeated droplet. Aspiration of the solution from the electrode grid.
Attach the cap to seal the MEA chamber for storage and future use. This procedure yields cortex and VTA sections for about 12 co-culture and should be performed inside a lamina flow hood under sterile conditions using healthy one to two days postnatal animals, total time for tissue collection should be less than one hour. After decapitation begin brain removal by first making two lateral scissor cuts to remove the skin from the scalp.
Remove then cut the skull open using fine eye scissors, making one sagittal midline cut on one coronal cut at the cortex cerebellum junction. Flip back all four of these skull flaps to expose the brain. Next with a sharpened spatula, cut frontally through the olfactory bulb.
Then advance the spatula quarterly underneath the brain. Gently lift the brain outta the skull and let it slide into chilled gaze. Solution for rapid cooling and temporary storage.
Repeat the above procedure for two more brains. To obtain VTA tissue, transfer the brains onto a sterile dry Petri dish. Using a small spatula, remove any excess fluid by gently sliding each brain sideways about one centimeter.
Then use a razor blade to remove the brainstem by making a coronal vertical cut at the level of the cerebellum. Now glue an agar block onto a mounting disc for mechanical stabilization of the brains during the slicing procedure. Then place a thin line of super glue, a few millimeters in front of the agar block on the disc, but avoid having the glue touch the agar using a small spatula.
Transfer and mount each brain frontal pole down. Ensure that the frontal poles are glued to the mounting disc and that the ventral sides touch the agar without any glue residue. This assures proper mechanical stabilization during cutting and easy lift off of cut slices.
Next, carefully submerge and secure the mounting disc with the mounted brains in a vibram tray filled with chilled gaze solution. Then with a carefully cleaned razor blade cut, 400 micrometer thick coronal sections of the midbrain at the highest vibration frequency and relatively low forward speed using an inverted pasta pipette with suction bulb, collect and transfer the slices containing the VTA into 35 by 10 millimeter Petri dishes filled with sterile chilled gaze solution for cortical sections. Repeat the previous slicing steps, but apply a vertical cut between the cortex and cerebellum and mount the four brains with the frontal pole up.
Collect about three coronal slices of 350 micrometer thickness starting at the level of the striatum. Now using a micro knife made of broken razor blades, dissect about two millimeter wide coronal sections of frontal cortex and midbrain areas containing the VTA under a stereo microscope. Collect the tissue section separately in small dishes filled with chilled gaze solution.
To begin mounting the tissue in the MEA chamber first position, the MEA at room temperature under a stereo microscope with the electrode array in focus. Then center a 25 microliter droplet of plasma on the clean dust-free and sterile electrode array matrix. Using a small spatula carefully slide a cortex and VTA section into the plasma droplet.
Now place the MEA on a cooling plate and refocus the view. Allow this to chill for about 15 seconds before adding 25 microliters of thrombin into the plasma droplet. Then using the thrombin pipette tip, carefully spread the plasma thrombin mixture with small circular movements across the MEA.
Be careful not to touch the brittle electrode array directly. Next, gently position the cortex on the array with the dorsal border along the second electrode row of the array. This way, the developing superficial layers will eventually cover the remainder of the array.
Then place the VTA adjacent to the ventral border of the cortex section. Now cap and loosely close the MEA chamber to retain high humidity. Allow the MEA culture assembly to sit for about six minutes inside the hood at room temperature to allow for plasma thrombin, coagulation, elation.
Meanwhile, repeat the procedure to prepare three more cultures. Next, in small droplets carefully add 600 microliters of culture medium to the culture chambers, using a one cc syringe with a 25 by five eighth gauge needle. Then tightly close the MEA chamber and place the MEA culture assembly onto the rocking storage tray inside the incubator.
After two days, in vitro, add 10 microliters of mitosis inhibitor. Then refresh the culture media by 60%at four days in vitro and every four days thereafter. During normal growth, the culture will flatten substantially and expand slightly dorsally.
Due to the development of superficial cortical layers, a healthy culture is identified by its opaque grayish tissue in brightfield. Conversely, bright reflective vesicles, a characteristic of dead degenerative cells about 10 to 12 days after preparing the cultures, they're ready for recording and stimulation. To begin a recording session, the MEA is transferred from the storage tray to the recording head stage.
To record local field potential or LFP, use a band pass filter of one to 200 hertz. Multiunit activity can also be studied using a band pass filter of 300 to 3000 hertz. Healthy spontaneous activity tends to occur in bursts of diverse size and duration.
To study the relationship between LFP and unit activity, we may calculate spike triggered averages of LFP. For these cortex cultures, negative LFP deflections are correlated with neuronal spiking to record stimulus evoked activity. First, the temporal waveform and amplitude of stimuli are specified with software which controls a stimulus generator.
A useful stimulation paradigm is the current controlled charge neutral single shock with bipolar square waveform. Next, an electrode is selected, which will be used to deliver the stimuli. Typical stimulus evoked LFP responses appear similar to spontaneous activity bursts.
Blanking circuitry disconnects the amplifiers during stimulation to reduce stimulus artifacts and to prevent amplifier saturation. The stimulation electrode requires several seconds to recover from stimulation and thus cannot be used to record response activity. On the MEA tends to emerge in spatiotemporal clusters with activity at one electrode, often accompanied by activity at other sites.
At the same time, typical waveforms of the LFP during such activity periods are shown here by over plotting three clusters occurring several seconds apart for each cluster. Negative LFP deflections can be seen at several electrodes within a window of one second when extracting NLFP peaks that cross a threshold of multiple negative sd, the activity in the form of NLFP peak times is conveniently visualized in a Rasta in which columns of dots represent near coincident NPS at various electrodes. The spatiotemporal organization of this activity is rather complex.
Columns which appear more or less homogeneous at low temporal resolution are composed of separate clusters at higher temporal resolution and so on. The emergence of space, GEOTEMPORAL and LFP clusters is highly organized in cortical networks. More specifically, the organization is scale in variant for neuronal avalanches.
This is demonstrated by calculating the probability of cluster sizes at a given temporal resolution. Delta T.Here clusters are composed of NL FPS that occur in the same or successive time bins when the size of such a cluster is expressed in total number of NFPS per cluster or integrated NLFP amplitudes per cluster, the cluster size distributions reveals a power law with an exponent close to minus 1.5. After watching this video, you should have a good understanding of how to carefully dissect brain tissue and grow cultures on multi electrode arrays in order to study the self-organization of neuronal activity.
These methods have also been used to study the relationship between spontaneous activity and stimulus evoked activity in cortical networks.
