This method can help answer key questions in the study of seizure mechanisms, such as, what neural subpopulations are responsible for electrographic seizure onset and termination. The main advantage of this technique, is that it can reproduce electrographic seizure events, on demand, and in vivo and in vitro models that are reminiscent of those observed clinically. This technique can also be used to search for potential anti-seizure therapies, by attempting to trigger electrographic seizure events during the application of different anti-seizure drug candidates.
To prepare the cortical slices, glue the preclinical brain tissue onto the Vibratome stage, using instant adhesive glue. Gently place the specimin holder into the buffer tray. Insure that the dorsal portion of the brain is facing the Vibratome's blade.
Slice the brain into 450 micrometer thick sections, in the dorsal to ventral direction. Make the first cut in the preclinical animal brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory motor area is observed.
Use a wide bore pipette. Transfer the targeted coronal slices to a Petrie dish containing cold dissection solution. For the coronal slices, make a transverse cut just below the neo cortical commissure, and then, cut at the midline to separate the hemispheres.
Then, use a new razor blade to cut off any excess tissue from the slices. It's critical to perform the brain-slicing procedure efficiently. Every moment the brain tissue is not submerged in ACSF, is detrimental to its quality.
Damaged, poor quality brain tissue is less likely to generate electrographic seizure events. Transfer the dorsal portion of the coronal slices, that contain the neo cortex, to a second Petrie dish filled with 35 degree Celsius ACSF, for a moment. Then, promptly transfer the slices to an incubation chamber, containing 35 degrees Celsius carbogenated ACSF.
Leave the brain slices slightly submerged in the incubation chamber at 35 degrees Celsius for 30 minutes. Then, remove the incubation chamber from the water bath and allow it to return to room temperature. Wait one hour for the brain slices to recover, before performing electrophysiological recordings.
In this procedure, cut out lens paper that is slightly larger than the brain slice. Use a wide-bore pipette or a detailing brush to transfer a brain slice onto the cut out lens paper that is held in place using a dental tweezer. Then, transfer the lens paper with a brain slice to the recording chamber, and secure it into position with a harp screen.
Subsequently, profuse the brain slice in the recording chamber with carbogenated ACSF at 35 degrees Celsius at a rate of three milliliters per minute. Use a digital thermometer to ensure the recording chamber is 33 to 36 degrees Celsius. Then, backfill the glass electrodes with 10 microliters of ACSF, using a Hamilton syringe.
Under the 20 times stereomicroscope, guide the recording glass electrode into the superficial cortical layer, two, three, using manual manipulators. View the electrical activity of the brain slice with standard software. To induce electrographic seizure-like activities, profuse the brain slice with ACSF containing 4-AP at 100 micromolar.
View the electrical activity of the brain slice with standard software. To generate electrographic seizure-like events, using optogenetic strategy on the brain slices from optogenetic mice, use a manual manipulator to position a 1, 000 micron cord diameter optical fiber directly above the recording region. Apply a brief pulse of blue light to initiate an ictal event.
A user-friendly MATLAB based program was specifically developed to detect and classify the various types of epileptiform events that occur in the in vitro and in vivo 4-AP seizure models. This detection program is available from the Valiante Labs GitHub repository. The application of 100 micromolar 4-AP to good quality 450 micron-sized cortical brain slices from a juvenile VGAT channelrhodopsin mouse, reliably induced recurrent ictal-like events within 15 minutes.
The application of 100 micromolar 4-AP to slices of poor quality, resulted in bursting events or spiking activity. On average, 40%of the slices from each dissected preclinical brain, successfully generated ictal-like events. Moreover, 83%of the dissected mice resulted in at least one brain slice that successfully generated ictal-like events.
In brain slices with spontaneously occurring ictal-like events, the application of a brief 30 millisecond light pulse on the brain slice, reliably triggered an ictal-like event that was identical in morphology. The same findings were made in brain slices from Thy one Channelrhodopsin two mice. Thus, regardless of which neuronal subpopulation was activated, any brief synchronizing event in the isolated cortical neural network, led to the onset of an ictal-like event.
These ictal-like events were comprised of a sentinel spike, tonic-like firing, clonic-like firing, and burst-like activity toward the end. They were similar in nature to the electrographic signatures associated with clinical seizures. Following this procedure, other types of brain state transitions can be performed to address questions like, what are the neuro biological mechanisms underlying brain state transitions?
And how can we regulate these transitions to prevent entry into various pathological brain states?
