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

Podsumowanie

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.

Streszczenie

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 "no seizure" 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 α-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.

Wprowadzenie

Electroconvulsive therapy has been used to treat patients with major depressive disorders, including severe drug-resistant depression, bipolar depression, Parkinson's diseases, and schizophrenia^1,2. In this therapy, seizure is generated by electrical stimulus delivered to the head of anesthetized patients via epicranial electrodes^1,2,3. Repetitive administration of ECS has been clinically beneficial to drug-resistant depressive disorders^1,2,3. 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 network^4,5, 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 hippocampus^7,8,9.

In ECS models, electrical stimulation is delivered to rodents via stereotaxically implanted electrodes, corneal electrodes, or ear electrodes to evoke generalized tonic-clonic seizures^10,11. Stereotaxic implantation of electrodes involves brain surgery and requires significant time to improve the experimenter'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 PSD^12,13,14. The PSD is a dense and well-organized subcellular domain in neurons in which synaptic proteins are highly concentrated at and near the postsynaptic membrane^12,13,15. 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 PSD^12,15,16,17 are correlated with synaptic plasticity and the synaptopathy observed in several neurological disorders^17,18. 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 gradients^14,19. The major challenge with this traditional method is that it requires large amounts of rodent brains^14,19. 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 centrifugation^20,21, 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 µg 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 α-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.

Protokół

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

1. 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.
2. Wean the rat pups at postnatal day (P) 28 and house them in groups of 2 - 4 male or female littermates.
3. Mark the tails of male rats with a non-toxic permanent black marker for identification.
4. Weigh the male rats 3 times per week and record their bodyweights.

2. Preparation of an ECS Machine

1. At 7:30 am, disinfect the bench in the animal preparation room and place an ECS machine (pulse generator) on the bench.
2. Place an individual male rat weighing 200 - 250 g in a clean, empty cage with a lid. Repeat this for all male rats to be treated with ECS induction. Let the rats habituate for 30 min.
3. While the rats are habituating in their cages, set a pulse generator for ECS induction to the frequency of 100 pulses/s, a pulse width of 0.5 ms, a shock duration of 0.5 s, and a current of 55 mA (Figure 1A).
4. Prepare the pulse generator by pushing the "RESET" button and ensuring that the "READY" button is lit. Make sure that the ear clips are not attached to a pulse generator and then press the "SHOCK" button for a few seconds.
   NOTE: At this point, the pulse generator is ready for ECS induction.
5. Plug the ear clips into the pulse generator.

3. Induction of Acute ECS

NOTE: See Figure 1B, top panel.

1. Wet the ear clips with sterile saline and ensure that they are saturated.
2. Wet a rat's ears with sterile saline by wrapping them in saline-soaked gauze. Once they are wet, remove the gauze.
3. Attach one clip per ear, position beyond the main cartilage band.
4. Confirm on the ECS machine that a true loop is established; if not, an error message or a reading of "1" will appear on the machine.
5. Wear a thick, non-metal glove. While holding the rat gently in a gloved hand, press the "SHOCK" button for a few seconds and slowly release the grip on the rat to observe the seizure. For the sham "no seizure" (NS) control, handle the rat identically but do not deliver the current.
6. Disconnect the ear clips as clonus begins and record the seizure behavior according to a revised Racine's scale²² that includes "mouth and facial movements" (stage 1), "head nodding" (stage 2), "forelimb clonus" (stage 3), "rearing with forelimb clonus" (stage 4), and "rearing and falling with forelimb clonus" (stage 5). The seizure should last approximately 10 s; record the seizure duration using a timer.
7. Following seizure termination, return the rat to its home cage. Monitor the rat for another 5 min to make sure of the recovery of the rat from the seizures. Keep it singly housed in the cage and return the cage to the recovery room.
8. Repeat the ECS induction on the next rat.
9. Monitor the rats throughout the remainder of the day and at least once in the morning and once in the afternoon the following day until they are euthanized for experiments.
   NOTE: The ECS induction method could lead to clinical signs as an incidental side effect, which requires attention. For example, the ECS induction protocol could induce seizures that last longer than 15 s and cause unnecessary distress to the rats. In this case, terminate the seizure using diazepam (10 mg/kg, i.p.) or pentobarbital (25 - 30 mg/kg, i.p.). If the rats develop respiratory distress or severe behavioral abnormalities following seizure cessation, euthanize them by carbon dioxide inhalation followed by decapitation.

4. Induction of Chronic ECS

NOTE: See Figure 1B, bottom panel.

1. Induce one ECS per day at the same time in the morning as described in steps 1 - 3, above, for seven consecutive days.
2. Monitor the rats twice a day after they are returned to their home cages.

5. Homogenization and Fractionation of Rat Hippocampi

NOTE: See Figure 2.

1. Prepare a fresh homogenization buffer (Solution A) that contains 320 mM sucrose, 5 mM sodium pyrophosphate, 1 mM EDTA, 10 mM HEPES pH 7.4, 200 nM okadaic acid, and protease inhibitors. Filter-sterilize the buffer using filters with a 0.22 µm pore size for vacuum filtration and place it on ice.
2. At a given time point following acute ECS or chronic ECS (Figure 1), euthanize the rat by CO₂ inhalation for 5 - 10 min, followed by decapitation using a guillotine.
3. Remove the brain and dissect the hippocampi on the metal plate placed on ice, as described previously^23,24.
4. Place two hippocampi from one rat onto a 30-mm tissue culture dish and mince them into small pieces using scissors.
5. Transfer the minced hippocampi to a manual glass homogenizer using a 1 mL pipette and add 1 mL of ice-cold homogenization buffer (Solution A, step 5.1). Insert a round pestle into a glass homogenizer. While the glass homogenizer is on ice, gently and steadily stroke up and down on the pestle 10 - 15 times for 1 min, until small pieces of hippocampal tissue disappear.
6. Transfer the homogenate to a 1.7 mL microcentrifuge tube using a 1-mL pipette and centrifuge the homogenate at 800 x g for 10 min at 4 °C to separate the postnuclear supernatant (S1 fraction) from the pellet containing insoluble tissue and nuclei (P1 fraction). Transfer 50 µL and 950 µL of the S1 fraction to two separate, new 1.7 mL microcentrifuge tubes using a 1 mL pipette and store these tubes on ice. Save the P1 fraction pellet on ice.
7. Centrifuge the S1 fraction (950 µL) for 10 min at 13,800 x g and 4 °C to separate the supernatant (S2 fraction), enriched with cytosolic-soluble proteins, and the pellet (P2 fraction), enriched with membrane-bound proteins, including synaptosomal proteins. Transfer the S2 fraction to a new 1.7 mL microcentrifuge using a 1-mL pipette and store it on ice.
8. Resuspend the pellet (P2 fraction) in 498 µL of ice-cold purified water using a 1-mL pipette. Add 2 µL of 1 M HEPES (pH 7.4) using a 20 µL pipette to achieve a final concentration of 4 mM HEPES (pH 7.4). Incubate at 4 °C with agitation for 30 min. Store the resuspended P2 fraction on ice.
9. Determine the protein concentration of the S1, S2, and P2 fractions using a BCA assay. Add 50 mM HEPES (pH 7.4) to each fraction to achieve a 1 mg/mL concentration and store at -80 °C until use, or process the P2 fraction to isolate the PSD.

6. Isolation of the PSD from the Crude Membrane Protein (P2) Fraction

1. Centrifuge the P2 fraction (500 µL) for 20 min at 25,000 x g and 4 °C to separate the lysed supernatant (LS2 fraction) and the lysed pellet (LP1 fraction). Transfer the LS2 fraction to a new 1.7 mL microcentrifuge tube using a 1 mL pipette and store it on ice.
2. Resuspend the LP1 pellet in 250 µL of 50 mM HEPES (pH 7.4) mixed with 250 µL of 1% detergent in 1x PBS buffer using a 1 mL pipette. Incubate at 4 °C with gentle agitation for 15 min.
3. Centrifuge the resuspended LP1 pellet for 3 h at 25,000 x g and 4 °C to separate the supernatant (non-PSD fraction) from the pellet (PSD fraction). Remove the supernatant to a 1.7-mL microcentrifuge tube and resuspend the PSD pellet in 100 µL of 50 mM HEPES (pH 7.4) using a 200 µL pipette.
4. Determine the protein concentration of the LS2, non-PSD, and PSD fractions using a BCA assay. Add 50 mM HEPES (pH 7.4) to each fraction to achieve a 1 mg/mL concentration and store at -80 °C until use.
   NOTE: All solutions used in steps 5 - 6 (i.e., Solution A, HEPES buffer, and PBS buffer) should be made with purified water free of ionic and organic contaminants and particles.

7. Western Blotting

1. Thaw each protein fraction on ice. Transfer 12 µL of each fraction (S2, P2, and PSD in 1 mg/mL) to a new 1.7 mL microcentrifuge tube using a 20-µL pipette.
2. Add 3 µL of 5x SDS sample buffer and incubate at 75 °C for 30 min in a water bath. Cool the sample down to room temperature (RT).
3. Load 10 µL of the protein sample into each well of 4-20% gradient 15-well comb SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel using a 20 µL pipette. Run the gel in the SDS-PAGE apparatus and at 80-100 V in running buffer (25 mM Tris, 190 mM glycine, and 0.1% SDS; pH 8.3).
   NOTE: Each gel should contain protein samples from NS rats and ECS rats at different time points following acute or chronic ECS.
4. Transfer the proteins from the SDS-PAGE gel to a polyvinyl difluoride (PVDF) membrane in the transfer apparatus at 25 - 30 V (60 mA) for 9 - 12 h in transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol; pH 8.3).
5. Remove the PVDF membrane from the transfer apparatus and wash it in Tris-buffered saline (TBS) for 5 min on a multi-purpose rotator at RT.
6. Block the membrane in 5% milk and 0.1% Tween-20 in TBS for 1 h. Incubate it in primary antibodies (Table 1) in washing buffer (1% milk and 0.1% Tween-20 in TBS) overnight on a multi-purpose rotator at 4 °C (see the Table of Materials for dilutions).
7. Wash the membrane 4 times for 10 min each in wash buffer and then incubate it with horseradish peroxidase (HRP)-conjugated secondary antibodies in washing buffer for 1 h on a multi-purpose rotator at RT.
8. Wash the membrane 4 times for 10 min each in wash buffer and then with TBS for 5 min.
9. Incubate the membrane with enhanced chemifluorescence substrate for 1 min and expose it to X-ray film. Develop the exposed film with a film processor.

8. Quantification of Western Blots

1. Scan the Western blot as a TIFF file and save this file to the computer.
2. Open the Western blot file in the ImageJ program as a grayscale image, under "File," "Open image," right arrow "gray-scale."
3. Choose the rectangular selection tool from the ImageJ toolbar and draw a rectangle that covers a single Western blot band of a protein of interest.
4. Under "Analyze," hit "Measure" to obtain the area and the mean density of a selected band.
5. Move the rectangle to a background area without changing its size and shape. Repeat step 8.4 to the area and the mean density of a background.
6. Subtract the mean density value of a background band from that of a Western blot band, giving the background-subtracted band density of the protein of interest.
7. Repeat steps 8.2 - 8.6 for all Western blot bands of interest.
8. Divide the background-subtracted band density of the protein of interest by the background-subtracted band density of a housekeeping gene product, such as α-Tubulin; this step yields the normalized value of the protein of interest.

Wyniki

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 "no seizure" rats did not receive an ...

Dyskusje

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 humans^1,2,3. Despite use of electroconvulsive therapy to treat severe depression, the precise underlying mechanism remains unclear. Because ECS induces anti-depressant-like beha...

Materiały

Name	Company	Catalog Number	Comments
Spargue-Dawley rat	Charles River Laboratories		ECS supplies
A pulse generator	Ugo Bsile, Comerio, Italy	57800	ECS supplies
MilliQ water purifying system	EMD Millipore	Z00Q0VWW	Subcellular fractionation supplies
Sucrose	Em science	SX 1075-3	Subcellular fractionation supplies
Na4O7P2	SIGMA-ALDRICH	221368	Subcellular fractionation supplies
Ethylenediaminetetraacetic acid (EDTA)	SIGMA-ALDRICH	E9884	Subcellular fractionation supplies
HEPES	SIGMA-ALDRICH	H0527	Subcellular fractionation supplies
Okadaic acid	TOCRIS	1136	Subcellular fractionation supplies
Halt Protease Inhibitor	Thermo Scientific	78429	Subcellular fractionation supplies
NaVO3	SIGMA-ALDRICH	72060	Subcellular fractionation supplies
EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters	Fisher Scientific	SCGPS05RE	Subcellular fractionation supplies
Iris Scissors	WPI (World Precision Instruments)	500216-G	Subcellular fractionation supplies
30 mm tissue culture dish	Fisher Scientific	08-772B	Subcellular fractionation supplies
Glass homogenizer and a Teflon pestle	VWR	89026-384	Subcellular fractionation supplies
1.7 mL microcentrifuge tube	DENVILLE SCIENTIFIC INC.	C2170 (1001002)	Subcellular fractionation supplies
Sorvall Legend XT/XF Centrifuge	Thermo Fisher	75004521	Subcellular fractionation supplies
Pierce BCA Protein Assay Reagent A, 500 mL	Thermo Fisher	#23228	Western blot supplies
Pierce BCA Protein Assay Reagent B, 25 mL	Thermo Fisher	#1859078	Western blot supplies
SDS-polyacrylamide gel (SDS-PAGE)	BIO-RAD	#4561086S	Western blot supplies
Running Buffer	Made in the lab		Western blot supplies.
Mini-PROTEAN Tetra Vertical Electrophorsis Cell for MiniPrecast Gels, 4-gel	BIO-RAD	#1658004	Western blot supplies
Polyvinyl difluoride (PVDF) membrane	Milipore	IPVH00010	Western blot supplies
Transfer Buffer	Made in the lab		Western blot supplies.
Tris-base	Fisher Scientific	BP152-1	Western blot supplies
Glycine	Fisher Scientific	BP381-5	Western blot supplies
Sodium dodecyl sulfate	SIGMA-ALDRICH	436143	Western blot supplies
Methanol	Fisher Scientific	A454-4	Western blot supplies
Triton X-100	Fisher Scientific	BP151-500	detergent for PSD isolation
Mini Trans-Blot Module	BIO-RAD	#1703935	Western blot supplies
Nonfat instant dry milk	Great value		Western blot supplies
Multi-purposee rotator	Thermo Scientific	Model-2314	Western blot supplies
Hyblot CL Autoradiography Film	DENVILLE SCIENTIFIC INC.	E3018 (1001365)	Western blot supplies
Enhanced chemifluorescence substrate	Thermo Scientific	32106	Western blot supplies
a Konica SRX-101A film processor	KONICA MINOLTA	SRX-101A	Western blot supplies
Name of Antibody
PSD-95	Cell Signaling	#2507	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
Synaptophysin	Cell Signaling	#4329	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
alpha-Tubulin	Santacruz	SC-5286	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
GluN2B	Neuromab	75-097	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
GluA2	Sigma-aldrich	Sab 4501295	Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Rabbit
STEP	Santacruz	SC-23892	Antibody dilution = 1:200 - 500, time = 9 - 12 h, Reaction Temperature = 4 °C, Host Species = Mouse
Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L)	Jackson ImmunoReserch laboratory	715-035-150	Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey
Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson ImmunoReserch laboratory	711-035-152	Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey