This study investigates the electrophysiological characteristics of organotypic brain slices from rat pups, focusing on the mechanisms underlying epilepsy. By simulating serum deprivation-induced stress, the research aims to elucidate the relationship between excitatory neurotransmitter excess and recurrent seizures.
Chemical neurotransmitters released from a neuron affect neighboring cells, allowing electrical impulses to pass between neurons. Excess of excitatory neurotransmitters cause disturbances to the brain's electrical activity, leading to recurrent seizures — abnormal repetitive neuron excitation and gradual neuronal death, leading to epilepsy.
To record epileptiform activity ex vivo, begin by taking a membrane insert containing rat pup brain-derived, rhinal cortex-hippocampus organotypic slices, which depict evolving epileptic events. Each slice has a defined dentate gyrus — DG region and cornu ammonis — CA region, consisting of tightly-packed multipolar pyramidal neurons.
Culture the slices for a prolonged duration, simulating gradual serum deprivation-induced stress conditions which lead to neuronal death, resembling in vivo epileptic conditions. Place one organotypic slice in the recording chamber of an electrophysiology — EEG — set up, with the hippocampus touching the bottom of the chamber.
The recording chamber is prefilled with a medium containing L-glutamine — a neurotransmitter precursor — that initiates neuronal excitation. Place the receiving electrode attached to a glass capillary containing artificial cerebrospinal fluid into a pyramidal neuron-rich CA3 region.
Record the electrical activity. Generate an electrograph, which briefly shows ictal events — areas with sudden peaks — corresponding to the beginning of epileptiform activity.
An increased number of ictal events, lasting for prolonged periods, indicates recurrent seizures.
Prepare the electrophysiology setup in a closed circuit. Verify that the flow rate of the interface-type chamber is set to two milliliters per minute, and open the carbox valve. Check the water level in the system, and place a piece of filter paper into the interface recording chamber to drain any excess medium.
Place a piece of lens-cleaning cleaning paper beneath the frame to supply medium to the slice, and turn on the temperature controller, amplifiers, and micromanipulators.
Use a syringe to load the glass electrode with freshly-prepared artificial cerebrospinal fluid. Place the glass electrode into the receiving electrode, and wait for the temperature in the interface chamber to stabilize at 37 degrees Celsius.
Working in a biosafety cabinet, place the insert in a 60-millimeter plate with a drop of medium. Use a sharp blade to cut a slice from the insert, and place the slice in the interface chamber with the hippocampus to the bottom right. Then, place the receiving electrode into the CA3 pyramidal cell layer.
Initiate the continuous acquisition protocol, and record the slice for 30 minutes.
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