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 female littermates.</li>
	<li>Mark the tails of male rats with a non-toxic permanent black marker for identification.</li>
	<li>Weigh the male rats 3 times per week and record their bodyweights.</li>
</ol>
2. Preparation of an ECS Machine
<ol>
	<li>At 7:30 am, disinfect the bench in the animal preparation room and place an ECS machine (pulse generator) on the bench.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Prepare the pulse generator by pushing the &#34;RESET&#34; button and ensuring that the &#34;READY&#34; button is lit. Make sure that the ear clips are not attached to a pulse generator and then press the &#34;SHOCK&#34; button for a few seconds. 
	NOTE: At this point, the pulse generator is ready for ECS induction.</li>
	<li>Plug the ear clips into the pulse generator.</li>
</ol>
3. Induction of Acute ECS
NOTE: See Figure 1B, top panel.
<ol>
	<li>Wet the ear clips with sterile saline and ensure that they are saturated.</li>
	<li>Wet a rat&#39;s ears with sterile saline by wrapping them in saline-soaked gauze. Once they are wet, remove the gauze.</li>
	<li>Attach one clip per ear, position beyond the main cartilage band.</li>
	<li>Confirm on the ECS machine that a true loop is established; if not, an error message or a reading of &#34;1&#34; will appear on the machine.</li>
	<li>Wear a thick, non-metal glove. While holding the rat gently in a gloved hand, press the &#34;SHOCK&#34; button for a few seconds and slowly release the grip on the rat to observe the seizure. For the sham &#34;no seizure&#34; (NS) control, handle the rat identically but do not deliver the current.</li>
	<li>Disconnect the ear clips as clonus begins and record the seizure behavior according to a revised Racine&#39;s scale22 that includes &#34;mouth and facial movements&#34; (stage 1), &#34;head nodding&#34; (stage 2), &#34;forelimb clonus&#34; (stage 3), &#34;rearing with forelimb clonus&#34; (stage 4), and &#34;rearing and falling with forelimb clonus&#34; (stage 5). The seizure should last approximately 10 s; record the seizure duration using a timer.</li>
	<li>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.</li>
	<li>Repeat the ECS induction on the next rat.</li>
	<li>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.</li>
</ol>
4. Induction of Chronic ECS
NOTE: See Figure 1B, bottom panel.
<ol>
	<li>Induce one ECS per day at the same time in the morning as described in steps 1 - 3, above, for seven consecutive days.</li>
	<li>Monitor the rats twice a day after they are returned to their home cages.</li>
</ol>
5. Homogenization and Fractionation of Rat Hippocampi
NOTE: See Figure 2.
<ol>
	<li>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 &#181;m pore size for vacuum filtration and place it on ice.</li>
	<li>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.</li>
	<li>Remove the brain and dissect the hippocampi on the metal plate placed on ice, as described previously23,24.</li>
	<li>Place two hippocampi from one rat onto a 30-mm tissue culture dish and mince them into small pieces using scissors.</li>
	<li>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.</li>
	<li>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 &#176;C to separate the postnuclear supernatant (S1 fraction) from the pellet containing insoluble tissue and nuclei (P1 fraction). Transfer 50 &#181;L and 950 &#181;L 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.</li>
	<li>Centrifuge the S1 fraction (950 &#181;L) for 10 min at 13,800 x g and 4 &#176;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.</li>
	<li>Resuspend the pellet (P2 fraction) in 498 &#181;L of ice-cold purified water using a 1-mL pipette. Add 2 &#181;L of 1 M HEPES (pH 7.4) using a 20 &#181;L pipette to achieve a final concentration of 4 mM HEPES (pH 7.4). Incubate at 4 &#176;C with agitation for 30 min. Store the resuspended P2 fraction on ice.</li>
	<li>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 &#176;C until use, or process the P2 fraction to isolate the PSD.</li>
</ol>
6. Isolation of the PSD from the Crude Membrane Protein (P2) Fraction
<ol>
	<li>Centrifuge the P2 fraction (500 &#181;L) for 20 min at 25,000 x g and 4 &#176;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.</li>
	<li>Resuspend the LP1 pellet in 250 &#181;L of 50 mM HEPES (pH 7.4) mixed with 250 &#181;L of 1% detergent in 1x PBS buffer using a 1 mL pipette. Incubate at 4 &#176;C with gentle agitation for 15 min.</li>
	<li>Centrifuge the resuspended LP1 pellet for 3 h at 25,000 x g and 4 &#176;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 &#181;L of 50 mM HEPES (pH 7.4) using a 200 &#181;L pipette.</li>
	<li>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 &#176;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.</li>
</ol>
7. Western Blotting
<ol>
	<li>Thaw each protein fraction on ice. Transfer 12 &#181;L of each fraction (S2, P2, and PSD in 1 mg/mL) to a new 1.7 mL microcentrifuge tube using a 20-&#181;L pipette.</li>
	<li>Add 3 &#181;L of 5x SDS sample buffer and incubate at 75 &#176;C for 30 min in a water bath. Cool the sample down to room temperature (RT).</li>
	<li>Load 10 &#181;L of the protein sample into each well of 4-20% gradient 15-well comb SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel using a 20 &#181;L 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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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 &#176;C (see the Table of Materials for dilutions).</li>
	<li>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.</li>
	<li>Wash the membrane 4 times for 10 min each in wash buffer and then with TBS for 5 min.</li>
	<li>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.</li>
</ol>
8. Quantification of Western Blots
<ol>
	<li>Scan the Western blot as a TIFF file and save this file to the computer.</li>
	<li>Open the Western blot file in the ImageJ program as a grayscale image, under &#34;File,&#34; &#34;Open image,&#34; right arrow &#34;gray-scale.&#34;</li>
	<li>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.</li>
	<li>Under &#34;Analyze,&#34; hit &#34;Measure&#34; to obtain the area and the mean density of a selected band.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat steps 8.2 - 8.6 for all Western blot bands of interest.</li>
	<li>Divide the background-subtracted band density of the protein of interest by the background-subtracted band density of a housekeeping gene product, such as &#945;-Tubulin; this step yields the normalized value of the protein of interest.</li>
</ol>

The authors declare that they have no competing financial interests.

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.

<table><tbody><tr><td>Spargue-Dawley rat</td><td>Charles River Laboratories</td><td></td><td>ECS supplies</td></tr><tr><td>A pulse generator</td><td>Ugo Bsile, Comerio, Italy</td><td>57800</td><td>ECS supplies</td></tr><tr><td>MilliQ water purifying system</td><td>EMD Millipore</td><td>Z00Q0VWW</td><td>Subcellular fractionation supplies</td></tr><tr><td>Sucrose</td><td>Em science</td><td>SX 1075-3</td><td>Subcellular fractionation supplies</td></tr><tr><td>Na&lt;sub&gt;4&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;P&lt;sub&gt;2&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>221368</td><td>Subcellular fractionation supplies</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>SIGMA-ALDRICH</td><td>E9884</td><td>Subcellular fractionation supplies</td></tr><tr><td>HEPES</td><td>SIGMA-ALDRICH</td><td>H0527</td><td>Subcellular fractionation supplies</td></tr><tr><td>Okadaic acid</td><td>TOCRIS</td><td>1136</td><td>Subcellular fractionation supplies</td></tr><tr><td>Halt Protease Inhibitor</td><td>Thermo Scientific</td><td>78429</td><td>Subcellular fractionation supplies</td></tr><tr><td>NaVO&lt;sub&gt;3&lt;/sub&gt;</td><td>SIGMA-ALDRICH</td><td>72060</td><td>Subcellular fractionation supplies</td></tr><tr><td>EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters</td><td>Fisher Scientific</td><td>SCGPS05RE</td><td>Subcellular fractionation supplies</td></tr><tr><td>Iris Scissors</td><td>WPI (World Precision Instruments)</td><td>500216-G</td><td>Subcellular fractionation supplies</td></tr><tr><td>30 mm tissue culture dish</td><td>Fisher Scientific</td><td>08-772B</td><td>Subcellular fractionation supplies</td></tr><tr><td>Glass homogenizer and a Teflon pestle</td><td>VWR</td><td>89026-384</td><td>Subcellular fractionation supplies</td></tr><tr><td>1.7 mL microcentrifuge tube</td><td>DENVILLE SCIENTIFIC INC.&amp;nbsp;</td><td>C2170 (1001002)</td><td>Subcellular fractionation supplies</td></tr><tr><td>Sorvall Legend XT/XF Centrifuge&amp;nbsp;</td><td>Thermo Fisher</td><td>75004521</td><td>Subcellular fractionation supplies</td></tr><tr><td>Pierce BCA Protein Assay Reagent A, 500 mL</td><td>Thermo Fisher</td><td>#23228</td><td>Western blot supplies</td></tr><tr><td>Pierce BCA Protein Assay Reagent B, 25 mL</td><td>Thermo Fisher</td><td>#1859078</td><td>Western blot supplies</td></tr><tr><td>SDS-polyacrylamide gel (SDS-PAGE)</td><td>BIO-RAD</td><td>#4561086S</td><td>Western blot supplies</td></tr><tr><td>Running Buffer</td><td>Made in the lab</td><td></td><td>Western blot supplies.&amp;nbsp;</td></tr><tr><td>Mini-PROTEAN Tetra Vertical Electrophorsis Cell for MiniPrecast Gels, 4-gel</td><td>BIO-RAD</td><td>#1658004</td><td>Western blot supplies</td></tr><tr><td>Polyvinyl difluoride (PVDF) membrane&amp;nbsp;</td><td>Milipore</td><td>IPVH00010</td><td>Western blot supplies</td></tr><tr><td>Transfer Buffer</td><td>Made in the lab</td><td></td><td>Western blot supplies.&amp;nbsp;</td></tr><tr><td>Tris-base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Western blot supplies</td></tr><tr><td>Glycine</td><td>Fisher Scientific</td><td>BP381-5</td><td>Western blot supplies</td></tr><tr><td>Sodium dodecyl sulfate</td><td>SIGMA-ALDRICH</td><td>436143</td><td>Western blot supplies</td></tr><tr><td>Methanol&amp;nbsp;</td><td>Fisher Scientific</td><td>A454-4</td><td>Western blot supplies</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151-500</td><td>detergent for PSD isolation</td></tr><tr><td>Mini Trans-Blot Module&amp;nbsp;</td><td>BIO-RAD</td><td>#1703935</td><td>Western blot supplies</td></tr><tr><td>Nonfat instant dry milk</td><td>Great value</td><td></td><td>Western blot supplies</td></tr><tr><td>Multi-purposee rotator&amp;nbsp;</td><td>Thermo Scientific</td><td>Model-2314</td><td>Western blot supplies</td></tr><tr><td>Hyblot CL Autoradiography Film</td><td>DENVILLE SCIENTIFIC INC.&amp;nbsp;</td><td>E3018 (1001365)</td><td>Western blot supplies</td></tr><tr><td>Enhanced chemifluorescence substrate&amp;nbsp;</td><td>Thermo Scientific</td><td>32106</td><td>Western blot supplies</td></tr><tr><td>a Konica SRX-101A film processor</td><td>KONICA MINOLTA</td><td>SRX-101A</td><td>Western blot supplies</td></tr><tr><td>&lt;strong&gt;Name of Antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PSD-95</td><td>Cell Signaling</td><td>#2507</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>Synaptophysin</td><td>Cell Signaling</td><td>#4329</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>alpha-Tubulin</td><td>Santacruz</td><td>SC-5286</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>GluN2B</td><td>Neuromab</td><td>75-097</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>GluA2</td><td>Sigma-aldrich</td><td>Sab 4501295</td><td>Antibody dilution = 1:500 - 1,000, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Rabbit</td></tr><tr><td>STEP</td><td>Santacruz</td><td>SC-23892</td><td>Antibody dilution = 1:200 - 500, time = 9 - 12 h, Reaction Temperature = 4 &amp;deg;C, Host Species = Mouse</td></tr><tr><td>Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoReserch laboratory</td><td>715-035-150</td><td>Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey</td></tr><tr><td>Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson ImmunoReserch laboratory</td><td>711-035-152</td><td>Antibody dilution = 1:2,000-5,000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey</td></tr></tbody></table>

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 ...

electroconvulsive-seizures-rats-fractionation-their-hippocampi-to

Research

JoVE Journal

Neuroscience

11.9K Views.  University of Illinois at Urbana-Champaign. 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.

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

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Medicine

Non-restraining EEG Radiotelemetry: Epidural and Deep Intracerebral Stereotaxic EEG Electrode Placement

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and neurodegenerative diseases as well as epilepsies. This powerful technique does not only provide valuable insights into the underlying pathophysiological mechanisms, i.e., the etiopathogenesis of CNS related diseases, it also facilitates the development of new translational, i.e., therapeutic approaches. Whereas competing techniques that make use of recorder systems used in jackets or tethered systems suffer from their unphysiological restraining to semi-restraining character, radiotelemetric EEG recordings overcome these disadvantages. Technically, implantable EEG radiotelemetry allows for precise and highly sensitive measurement of epidural and deep, intracerebral EEGs under various physiological and pathophysiological conditions. First, we present a detailed protocol of a straight forward, successful, quick and efficient technique for epidural (surface) EEG recordings resulting in high-quality electrocorticograms. Second, we demonstrate how to implant deep, intracerebral EEG electrodes, e.g., in the hippocampus (electrohippocampogram). For both approaches, a computerized 3D stereotaxic electrode implantation system is used. The radiofrequency transmitter itself is implanted into a subcutaneous pouch in both mice and rats. Special attention also has to be paid to pre-, peri- and postoperative treatment of the experimental animals. Preoperative preparation of mice and rats, suitable anesthesia as well as postoperative treatment and pain management are described in detail.

Non-restraining EEG radiotelemetry is a valuable methodological approach to record in vivo long-term electroencephalograms from freely moving rodents. This detailed protocol describes stereotaxic epidural and deep intracerebral electrode placement in different brain regions in order to obtain reliable recordings of CNS rhythmicity and CNS related behavioral stages.

Implantable EEG radiotelemetry is of central relevance in the neurological characterization of transgenic mouse models of neuropsychiatric and ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.

Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain ...

Electrophoretic Delivery of &#x3B3;-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the cases, serious side effects affect the quality of life of patients. Moreover, a high percentage of epileptic patients are drug resistant; in their case, neurosurgery or neurostimulation are necessary. Therefore, the major goal of epilepsy research is to discover new therapies which are either capable of curing epilepsy without side effects or preventing recurrent seizures in drug-resistant patients. Neuroengineering provides new approaches by using novel strategies and technologies to find better solutions to cure epileptic patients at risk.
As a demonstration of a novel experimental protocol in an acute mouse model of epilepsy, a direct in situ electrophoretic drug delivery system is used. Namely, a neural probe incorporating a microfluidic ion pump (&#181;FIP) for on-demand drug delivery and simultaneous recording of local neural activity is implanted and demonstrated to be capable of controlling 4-aminopyridine-induced (4AP-induced) seizure-like event (SLE) activity. The &#947;-aminobutyric acid (GABA) concentration is kept in the physiological range by the precise control of GABA delivery to reach an antiepileptic effect in the seizure focus but not to cause overinhibition-induced rebound bursts. The method allows both the detection of pathological activity and intervention to stop seizures by delivering inhibitory neurotransmitters directly to the epileptic focus with precise spatiotemporal control.
As a result of the developments to the experimental method, SLEs can be induced in a highly localized manner that allows seizure control by the precisely tuned GABA delivery at the seizure onset.

Electrophoretic Delivery of &#947;-aminobutyric Acid GABA into Epileptic Focus Prevents Seizures in Mice

The challenge of epilepsy research is to develop novel treatments for patients where classical therapy is inadequate. Using a new protocol&#8212;with the help of an implantable drug delivery system&#8212;we are able to control seizures in anesthetized mice by the electrophoretic delivery of GABA into the epileptic focus.

Epilepsy is a group of neurological disorders which affects millions of people worldwide. Although treatment with medication is helpful in 70% of the ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...

Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

<a href='https://app.jove.com/v/58455/microdialysis-excitatory-amino-acids-during-eeg-recordings-freely'>Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)</a>This video demonstrates the assessment of extracellular excitatory neurotransmitter levels in rats with chronic epilepsy induction. After implanting a microdialysis probe into the brain and confirming the absence of seizures using electroencephalography (EEG), extracellular fluid is collected to evaluate elevated neurotransmitter levels in epileptic rats. A high-potassium solution is then infused to sustain neuronal excitability and trigger an increased release of neurotransmitters, with samples collected to assess the potassium-induced peak in excitatory neurotransmitters.

Source: Soukupov&#225;, M., et al. Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats. J. Vis. Exp. (2018)This video ...

JoVE Encyclopedia of Experiments

Neuropathology

Epilepsy and Seizure Disorders

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app.jove.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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