Name: electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins
Uploaded: 2017-08-15T14:00:00
Description: Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

The authors thank Dr. Eric C. Bolton for allowing us to use his centrifuge for fractionation and Dr. Graham H. Diering in Dr. Richard L. Huganir's lab at John's Hopkins University for providing us with the small-scale protocol for the PSD fractionation.

All experimental procedures including animal subjects have been approved by the Institutional Animals Care and Use Committee at the University of Illinois at Urbana-Champaign.
1. Maintaining a Rat Colony
<ol>
	<li>Breed Sprague-Dawley rats (see the Table of Materials) and maintain them in standard conditions with a 12-h light-dark cycle and ad libitum access to food and water.</li>
	<li>Wean the rat pups at postnatal day (P) 28 and house them in groups of 2 - 4 male or femal...

Here, we describe an ECS induction method in rats that elicits the global stimulation of neuronal activity in their hippocampi. ECS is an animal model of electroconvulsive therapy, which is clinically used to treat drug refractory depressive disorders in humans1,2,3. Despite use of electroconvulsive therapy to treat severe depression, the precise underlying mechanism remains unclear. Because ECS induces anti-depressant-like beha...

Electroconvulsive therapy has been used to treat patients with major depressive disorders, including severe drug-resistant depression, bipolar depression, Parkinson&#39;s diseases, and schizophrenia1,2. In this therapy, seizure is generated by electrical stimulus delivered to the head of anesthetized patients via epicranial electrodes1,2,3. Repetitive administration of ECS has been clinically beneficial to drug-resistant depressive disorders1,2,3. However, the exact mechanism underlying the long-term efficacy of the antidepressant effect in humans has remained elusive. ECS is an animal model of electroconvulsive therapy and is widely used to investigate its therapeutic mechanism. In rodents, both acute ECS and chronic ECS treatment promote adult neurogenesis in the hippocampi and reorganize the neural network4,5, which is likely to contribute to improvements in cognitive flexibility. Furthermore, global elevation of brain activity by ECS alters the abundance of transcripts, such as a brain derived neurotropic factor6, and multiple proteins, including metabotropic glutamate receptor 17 and the N-methyl-D-aspartate (NMDA) type glutamate receptor subunits7. These changes are involved in mediating long-term modification of synapse number, structure, and strength in the hippocampus7,8,9.
In ECS models, electrical stimulation is delivered to rodents via stereotaxically implanted electrodes, corneal electrodes, or ear electrodes to evoke generalized tonic-clonic seizures10,11. Stereotaxic implantation of electrodes involves brain surgery and requires significant time to improve the experimenter&#39;s surgical skills to minimize injury. Less invasive corneal electrodes could cause corneal abrasion and dryness and require anesthesia. The use of ear-clip electrodes bypasses these limitations because they can be used on rodents without surgery or anesthesia and cause minimal injury. Indeed, we found that current delivered to awake rats via ear-clip electrodes reliably induces stage 4-5 tonic-clonic seizures and alters synaptic proteins in their hippocampi10.
To examine the ECS-induced abundance of synaptic proteins in the specific brain regions of the rodents, it is important to choose the experimental methods that are most suitable for their detection and quantification. Subcellular fractionation of the brain allows for the crude isolation of soluble cytosolic proteins; membrane proteins; organelle-bounds proteins; and even proteins in special subcellular structures, such as the PSD12,13,14. The PSD is a dense and well-organized subcellular domain in neurons in which synaptic proteins are highly concentrated at and near the postsynaptic membrane12,13,15. The isolation of the PSD is useful for the study of synaptic proteins enriched at the PSD, since dynamic changes in the abundance and function of postsynaptic glutamate receptors, scaffolding proteins, and signal transduction proteins in the PSD12,15,16,17 are correlated with synaptic plasticity and the synaptopathy observed in several neurological disorders17,18. A previous subcellular fractionation method used to purify the PSD involved the isolation of the detergent-insoluble fraction from the crude membrane fraction of the brain by the differential centrifugation of sucrose gradients14,19. The major challenge with this traditional method is that it requires large amounts of rodent brains14,19. Preparation of 10 - 20 rodents to isolate the PSD fraction per treatment requires extensive cost and time investment and is not practically feasible if there are many treatments.
To overcome this challenge, we have adapted a simpler method that directly isolates the PSD fraction, without sucrose gradient centrifugation20,21, and revised it to be applicable to PSD isolation from the hippocampi of a single rat brain.Our small-scale PSD fractionation method yields about 30 - 50 &#181;g of the PSD proteins from 2 hippocampi, sufficient for use in several biochemical assays, including immunoprecipitation and Western blotting. Western blotting demonstrates the success of our method for isolating the PSD by revealing the enrichment of postsynaptic density protein 95 (PSD-95) and the exclusion of presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin. Our ECS induction and small-scale PSD fractionation methods are easily adaptable to other rodent brain regions and provide a relatively simple and reliable way to evaluate the effects of ECS on the expression of PSD proteins.

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electroconvulsive seizures in rats and fractionation of their hippocampi to examine seizure-induced changes in postsynaptic density proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Using the detailed procedure presented here, one electrical shock (55 mA, 100 pulses/s for 0.5 s) delivered via ear-clip electrodes induced nonrecurring stage 4-5 tonic-clonic seizures in rats (Figure 1A-B). A total 8 of rats received acute ECS induction and displayed stage 4-5 tonic-clonic seizures. The seizures lasted about 10 s, and all rats recovered within 1 - 2 min of seizure cessation. Sham &#34;no seizure&#34; rats did not receive an ...

Watch this Scientific Journal Video about Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins at JoVE.com

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

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.

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