In the Parrish lab, we are keen on understanding how seizures start, propagate and terminate. We are particularly interested in exploring novel therapies for status epilepticus, a life-threatening condition in which a seizure does not self terminate. We use CMA's high density micro electrode array systems in our research.
These advanced technologies allow us to record high resolution electrophysiological data from brain slices, capturing detailed local field potentials. This helps us understand complex brain activities like seizure patterns with great spatial and temporal precision. In the future, we plan to explore the spatial and temporal propagation patterns of status epilepticus, a prolonged seizure state that often becomes resistant to anti-epileptic medication.
We plan to use the information from these studies to find more effective treatments for status epilepticus. Before starting chip preparation, designate transfer pipettes for various tasks. Fill the well of the Micro Electrode Array or MEA chip with 190 proof ethanol so that the bottom of the chip well is completely covered.
Let the ethanol sit for 30 to 60 seconds, then remove it with a discard pipette. Fill the well of the MEA chip with ACSF, and remove it with a discard pipette to rinse residual ethanol out of the chip well. Then add ACSF and let it stand for at least 30 seconds.
Before docking the MEA chip with an antistatic wipe with 190 proof ethanol, and use it to wipe the chip's pins, gently slide the MEA chip into the MEA platform, and engage the docking mechanism to lock the chip into place. Check the recording and reference electrodes for bubbles. If bubbles are present, take a clean paintbrush, and lightly sweep over the electrodes to remove them.
Check the chip for noise using the CMOS's HDMEA software, and visually scan the false color map for bubbles, non-biological oscillations or spikes caused by electrical interference. Ground the MEA system appropriately to negate any encountered noise. To begin, place a platinum harp in a weigh boat near the MEA platform.
Then cover the harp with about three milliliters of ACSF to reduce its hydrophobic tendencies. Use scissors to trim about 1.5 inches off the narrow tip of a transfer pipette. Next, use the modified pipette to collect a brain slice from the slice-holding chamber.
Gently dispense the brain slice, and any solution in the pipette into the chip well. To position the slice properly, use a soft paintbrush to create a current in the solution that pushes the brain slice onto recording electrodes. Using forceps, gently place the harp over the brain slice with the threads downwards to press the slice onto the recording electrodes.
Orient the harp so that the side without a frame faces toward the inflow needle, and the frame of the harp does not contact any of the recording electrodes. Now take a discard pipette, and remove XSACSF. Then take an antistatic wipe, twist a corner to create a tip, and use it to soak up the remaining ACSF surrounding the recording electrodes without touching the recording electrodes brain slice or harp.
Using a designated ACSF pipette, quickly add about two milliliters of carbogenated ACSF to cover the brain slice. Fill the well with about three milliliters carbogenated ACSF until it is roughly three quarters full. Then use a microscope or camera to take a high resolution picture of the brain slice on the MEA chip.
Place the inflow and outflow tubes into the beaker filled with ACSF. Place the inflow needle close to the bottom of the chip well just outside the recording electrodes. Then place the outflow needle close to the top of the chip well towards the edge so that the liquid rises almost to the brim of the chip well, about four milliliters.
Now set the perfusion inflow to five milliliters per minute, and the perfusion outflow to seven milliliters per minute. Turn on the inflow and outflow. Remove the inflow needle from the chip well until it begins to output solution instead of air.
After that, place the needle back inside the chip well. Then use a solution heater to keep the solution at or near physiological temperature around 34 to 37 degrees Celsius. Let the ACSF perfuse over the brain slice for 10 minutes.
After 10 minutes have elapsed, move the outflow tube to the discard beaker. Then move the inflow tube to the beaker containing the pro convulsant solution. Allow the non convulsant ACSF to be flushed out of the profusion system into the discard beaker for 10 minutes.
Finally, transfer the outflow tube into the beaker containing the pro convulsion solution, and allow it to cycle until the experiment finishes. Powerful electrographic seizure-like activity was frequently observed in the neocortical regions under both the zero magnesium, and 4-aminopurine paradigms. The hippocampal regions displayed more variability between brain slices with some demonstrating seizure-like activity, and others showing transient discharges.
Neocortical activity under the zero magnesium paradigm showed higher power in multiple frequency bands compared to the 4-aminopurine paradigm. Hippocampal activity in the zero magnesium paradigm demonstrated higher power in high gamma frequency bands compared to the neocortex in both brain regions in the 4-aminopurine paradigm.
