The overall goal of the electroconvlusive seizure induction and subsequent smallscale fractionation of hippocampus is to induce global seizure activity in the rat brain, and to examine seizure activity induced alterations of proteins in the postsynaptic density of the hippocampus. This method can help answer key questions in the neural process of the film, such as which snaptic proteins are regulated in specific brain regions upon seizure, and whether such regulation can contribute to neuroplasticity. The main advantage of this electroconvulsive seizure induction protocol is that we can lively induce lo-bal allevation of law-dons brain activity without neuronal death.
Demonstrating the procedure will be myself and Han Gil Jeong from Dr.Hee Jung Chung's laboratory. To begin this procedure, place a male rat in a clean, empty cage with a lid. Let the rat habituate for 30 minutes, then, set the pulse generator for ECS induction.
Prepare the pulse generator by pushing the reset button and ensure that the ready button is lit. Make sure that the ear clips are not attached to the pulse generator. Next, press the shock button for a few seconds, then, plug the ear clips into the pulse generator.
In this step, wet the ear clips with sterile saline and ensure that they are saturated. Next, wet the rat's ears with sterile saline by wrapping them in saline soaked gauze. Once they are wet, remove the gauze.
Attach the clips to the ears at one clip per ear and position them beyond the main cartilage band. Press the shock button for a few seconds and observe the seizure. For the sham, no seizure control, treat the rat identically, but do not deliver the current.
As clonus begins, disconnect the ear clips and record the seizure behavior according to a revised, Racine scale of one to five. This includes rearing with four limb clonus at stage four, and rearing and falling with four limb clonus at stage five. The seizure should last approximately 10 seconds.
Record the seizure duration using a timer. Following seizure termination, return the rat to its home cage. Monitor the rat for another five minutes to make sure of its recovery from the seizures.
Keep it singly housed in the cage, and return the cage to the recovery room. In this procedure, place two hippocampi from one rat onto a 30 milometer tissue culture dish and mince them into small pieces using scissors. After that, transfer the minced hippocampi to a manual glass homogenizer and add one milliliter of iced cold homogenization buffer.
Next, insert a round pecil into a glass homogenizer. While the glass homogenizer is on ice, gently and steadily stroke up and down on the pecil, 10 to 15 times for one minute until small pieces of hippocampal tissue disappear. Then, transfer the homogenote to a 1.7 milometer microcentrifuge tube using a one milometer pipet and centrifuge the homogenote at 800 times G for 10 minutes at four degrees Celsius to separate the post nuclear supernatant from the pellet containing insoluble tissue and nuclei.
Afterward, transfer 50 microliters and 950 microliters of the S1 fraction to two separate new 1.7 milliliter microcentrifuge tubes and store these tubes on ice. Also, save the P1 fraction pellet on ice. Subsequently, centrifuge the 950 microliter S1 fraction for 10 minutes at 13, 800 times G and four degrees Celsius to separate the supernatant enriched with cytosolic soluble proteins and the pellet enriched with membrane bound proteins, including synaptosomal proteins.
Transfer the S2 fraction to a new 1.7 milliliter microcentrifuge and store it on ice. Following that, resuspend the P2 fraction pellet in 498 microliters of ice cold purified water. Add two microliters of one molar heeps to achieve a final concentration of four millimolar heeps.
Incubate the suspension at four degrees Celsius with agitation for 30 minutes. After 30 minutes, store the resuspended P2 fraction on ice. In this step, centrifuge the P2 fraction for 20 minutes at 25, 000 times G and four degrees Celsius to separate the lysed supernatant and the lysed pellet.
Transfer the LS2 fraction to a new 1.7 milliliter microcentrifuge tube and store it on ice. Then, resuspend the LP1 pellet in 250 microliters at 50 millimolar heeps, mixed with 250 microliters of one percent detergent, and 1 X PBS. Incubate the suspension at four degrees Celsius with gentle agitation for 15 minutes.
Afterward, centrifuge the resuspended LP1 pellet for 3 hours at 25, 000 times G and four degrees Celsius to separate the non PSD fraction supernatant from the PSD fraction pellet. Remove the supernatant to a 1.7 milliliter microcentrifuge tube and resuspend the PSD pellet in 100 microliters of 50 millimolar heeps. In this figure, the representative western blots show the protein expression of NMDA receptive subunit GluN2B, ampa receptor, GluA2, and STEP 61 in the S2, P2, and PSD fractions from the hippocampi of sham, no seizure rats, and rats that received acute ECS.
The cytoplasmic soluble protein alpha-Tubulin is enriched in the S2 fraction. Synaptophysin is a presynaptic vesicle protein and is enriched in the crude membrane P2 fraction but not in the PSD fraction, whereas PSD-95 is enriched in both the P2 and PSD fractions. Shown here, are the quantification of GluN2B and GluA2 in the P2 and PSD fractions at three and 24 hours after acute ECS.
GluN2B and GluA2 expression in the P2 fraction was normalized to alpha-Tubulin in the S2 fraction. In GluN2B and GluA2 expression in the PSD fraction, was normalized to PSD 95 in the PSD fraction. Once mastered, this technique can be done in five to six hours if it is performed properly.
Following this procedure, auto-masser like mass, spector-metry, based on protein mix can be performed in order to answer additional questions, what other synaptic proteins are altered by ECS. After its development, this technique paved the way for researchers in the field of neurological disease to explore the underlying mechanism of synaptic dysfunction in order for act of law-dons, trans-gen-ing motors of neurological diseases. After watching this video, you should have a good understanding of how to induce the ECS in law-dons, perform homogenization and PSD fractionation from the hippocampi and to examine alterations of synaptic proteins after ECS.
