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 behaviors in rodents and stimulates hippocampal neurogenesis4,32, ECS has been extensively used to investigate if new adult-born neurons in the dentate gyrus of the hippocampus contribute to anti-depressant behavior5,32.
ECS induces mild to severe generalized tonic-clonic seizures in rodents upon current delivery through stereotaxically implanted electrodes, corneal electrodes, or ear-clip electrodes33,34. EEG recordings in patients have revealed that bilateral electrical stimulation causes more prominent and longer generalized seizures than unilateral stimulation does35,36. In our ECS induction method, tonic-clonic seizures are induced by current delivery through noninvasive ear-clip electrodes attached to both ears, mimicking seizures in humans evoked by bilateral stimulation36.&#160;Although we haven&#8217;t checked the efficacy of ECS in the rats to which sedation or anesthesia is applied, it is feasible that giving anesthetic drugs to the rats could reduce the degree of seizures induced by electrical stimulation, which might be due to the fact that anesthesia state is physiologically associated with enhanced inhibitory and dampened excitatory tone in the brain network. In addition, the critical step in the ECS induction method is to experimentally dial the current intensity to elicit stage 4 - 5 generalized tonic- clonic seizures based on the age, weight, and the species of rodent. &#160;Our ECS induction method has been optimized for inducing stage 4 - 5 seizures within a few seconds in awake male Sprague-Dawley rats weighing 200 - 250 g. If the rats under anesthesia state are used for ECS induction, the intensity, duration, and frequency of delivering current should be modified for successful induction of seizure ranging from stage 4 to 5.&#160;
ECS also offers the advantage of stimulating hyperactivity in neurons and causing acute transient seizures with very low mortality33, in contrast to chemoconvulsants, including pilocarpine and kainite, which induce status epilepticus and cause chronic manifestation of spontaneous recurrent seizures and severe histological alterations37,38. Although ECS has been routinely utilized to screen anti-epileptic drugs33,34, acute ECS and chronic ECS do not result in the generation of chronic epilepsy and thus cannot be used in an animal model to study epileptogenesis. Instead, ECS has been widely employed to examine the extent to which the broad elevation of brain activity alters expression and/or posttranslational modifications of synaptic proteins in vivo, which contribute to persistent changes in synaptic strength and structures (Figure 3 and Figure 4)10,40. In this study, we only used male rats to exclude the unexpected effects of the estrus cycle phase of female rats on the amount of PSD protein after ECS. However, it was observed that there is no sex difference in the scaffolding proteins, including PSD-95 and SAP102 in the PSD region of the frontal cortex and hippocampus39, implying that hormonal changes governing estrus cycle might not affect the basal amount of PSD proteins.
Multiple methods have been employed to examine the extent to which ECS induces changes in neurogenesis, synaptogenesis, and synaptic plasticity5,6,7,8,9,11,40. Electrophysiological recordings of excitatory postsynaptic current (EPSC) are widely used to detect the changes in the synaptic strength of glutamatergic excitatory synapses21. For example, miniature EPSC recordings have revealed the homeostatic downscaling of excitatory synaptic strength in the cortical pyramidal neurons of layers II-III of mice following acute ECS41. However, the identification of synaptic proteins that contribute to ECS-induced synaptic plasticity is challenging because electrophysiological recordings must be paired with the genetic knockout or knock-down of specific candidate synaptic proteins. Changes in the level of glutamate receptors at the postsynaptic membrane and synaptic proteins enriched in the PSD regulate the strength and efficacy of excitatory synaptic transmission17,18,25. Although immunohistochemistry can be used to examine ECS-induced changes in the expression of synaptic proteins, this technique can examine only 1 - 2&#160; candidate synaptic proteins at a time and requires well-validated antibodies that specifically recognize them without causing non-specific staining.
The subcellular fractionation of brain tissue in combination with Western blotting offers advantages over electrophysiology and immunohistochemistry in identifying synaptic proteins that are altered by ECS. The subcellular fractionation of brain tissue is a rapid and crude biochemical method to separate soluble cytosolic proteins (S2 fraction) from crude membranes (P2 fraction), including ER and Golgi membranes, membrane-bound organelles, plasma membranes, and synaptic terminal membranes that reseal to form synaptosomes42,43,44. The PSD fraction can be further isolated from the P2 fraction to enrich the synaptic proteins42,43,44. The unbiased proteomic analysis of the PSD fraction could identify all PSD proteins whose levels are altered by ECS. Western blotting can be performed rapidly to examine the expression changes of the PSD proteins, which can be easily identified from non-specific bands.
The previous method of brain fractionation requires a large amount of rodent brain tissue42,43,44, making this technique challenging for use when examining soluble or membrane proteins from an individual rodent brain or a specific brain region from a single brain. There is an increasing demand to quantitatively compare the proteome from one brain region to another and from a control animal to a transgenic animal or an animal that has undergone a specific treatment. Hence, we have revised and optimized the traditional brain fractionation method to isolate crude soluble and membrane fractions from two hippocampi of a single rat. Our small-scale crude fractionation protocol successfully isolated crude P2 membrane fractions from cytosolic S2 soluble fractions, as indicated by the lack of cytoplasmic protein &#945;-tubulin in the P2 fractions, whereas membrane-bound PSD95 was detected in the P2 but not S2 fractions (Figure 3A and Figure 4A). Using the method described here, we have quantitatively shown that acute ECS significantly increases STEP61 expression and decreases tyrosine phosphorylation of its substrates, GluN2B and the extracellular signal-regulated kinase 1/2 in the crude membrane P2 fraction of rat hippocampi at 48 h following acute ECS10.
Unexpectedly, the S2 fractions contained synaptophysin; STEP61; and, to a much lesser extent, GluA2 (Figure 3A and Figure 4A). STEP61 is a glycosylated integral membrane protein26 associated with the endoplasmic reticulum (ER) and PSD45. It is possible that the centrifugation to isolate the crude membrane pellet (P2 fraction) might not have been sufficient to pellet all synaptic vesicles containing synaptosomes, as well as endosomes and lysosomes containing GluA2 and STEP61. Nonetheless, clear enrichment of GluN2B, GluA2, and PSD95 and the lack of &#945;-tubulin in the P2 fractions indicate that our fractionation protocol can enrich membrane-bound proteins and transmembrane proteins that are not associated with synaptic vesicles and endosomes in the crude membrane P2 fraction.
Although the crude membrane P2 fraction contains synaptosomes, one limitation of examining activity-dependent alterations of synaptic proteins in the P2 fraction is that the exact location of their changes cannot be determined. If synaptic proteins enriched in the PSD were to be determined, then further biochemical fractionation can be used to isolate the PSD. The previous method of PSD fractionation requires a large amount of rodent brain tissue (i.e., 10 - 20 rodent brains) and sucrose gradients42,43,44. Because this traditional method is inadequate to isolate a sufficient amount of the PSD fraction from two hippocampi of a single rat, we have adapted a simpler method that directly isolates the PSD fraction, without a sucrose gradient20,21. This method yields about 30 - 50 &#181;g of the PSD protein, sufficient for several biochemical assays, including Western blotting. Our method enriches PSD95, GluN2B, and GluA2, which are known to be concentrated in the PSD fraction, and detects changes in GluN2B expression in PSD fraction following chronic ECS induction (Figure 3 and Figure 4).
Here, our quantitative Western blot analysis revealed no significant change in GluA2 expression in the P2 and PSD fractions at 3 h and 24 h following acute ECS (Figure 3C). The GluN2B expression was unaltered in the P2 fraction but displayed an increasing trend in the PSD fraction at 3 h and 24 h after acute ECS (Figure 3B), suggesting the possible differential regulation of synaptic versus extrasynaptic GluN2B-containing NMDA receptors. We also observed that GluN2B expression displayed a decreasing trend in the P2 fraction after chronic ECS and was significantly reduced in the PSD fraction at 24 and 96 h following chronic ECS (Figure 4B). Though highly speculative, such removal of GluN2B from the PSD following repetitive induction of ECS may be mediated by multiple mechanisms, including direct internalization from the PSD membrane or lateral diffusion to extrasynaptic sites. Considering our previous report that prolonged enhancement of neuronal activity markedly increases STEP61 expression and reduces Tyr-phosphorylation of GluN2B in the P2 fractions of cultured hippocampal neurons46, our findings suggest that a decrease in GluN2B expression in the P2 and the PSD fractions after chronic ECS may be likely due to enhanced STEP61 expression and the subsequent removal of GluN2B from the extrasynaptic membrane.
In summary, we have demonstrated that our small-scale PSD fractionation method, in combination with an ECS induction protocol, allows us to differentiate in vivo the activity-dependent regulation of glutamate receptors at the postsynaptic membrane versus total plasma membranes, including the extrasynaptic membranes. This protocol can easily be applied to any synaptic proteins at excitatory synapses and can be modified for other rodent hippocampi or other brain regions by adjusting the volume of each solution based on the tissue weight. Thus, our small-scale PSD fractionation method is versatile and can be adopted for future applications to examine the in vivo changes in the postsynaptic proteins in each animal upon genetic, pharmacological, or mechanical treatment.

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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			<td height="21" style="height:21px;width:433px;">Spargue-Dawley rat</td>
			<td style="width:245px;">Charles River Laboratories</td>
			<td style="width:145px;"></td>
			<td style="width:224px;">ECS supplies</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">A pulse generator</td>
			<td>Ugo Bsile, Comerio, Italy</td>
			<td>57800</td>
			<td>ECS supplies</td>
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		<tr height="21">
			<td height="21" style="height:21px;">MilliQ water purifying system</td>
			<td>EMD Millipore</td>
			<td>Z00Q0VWW</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="19" style="height:19px;">Sucrose</td>
			<td>Em science</td>
			<td>SX 1075-3</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="24" style="height:24px;">Na4O7P2</td>
			<td>SIGMA-ALDRICH</td>
			<td>221368</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="28" style="height:28px;width:433px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>SIGMA-ALDRICH</td>
			<td>E9884</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;">HEPES</td>
			<td>SIGMA-ALDRICH</td>
			<td>H0527</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;">Okadaic acid</td>
			<td>TOCRIS</td>
			<td>1136</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;">Halt Protease Inhibitor</td>
			<td>Thermo Scientific</td>
			<td>78429</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="25" style="height:25px;">NaVO3</td>
			<td>SIGMA-ALDRICH</td>
			<td>72060</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;width:433px;">EMD Millipore Sterito Sterile Vacuum Bottle-Top Filters</td>
			<td>Fisher Scientific</td>
			<td>SCGPS05RE</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;width:433px;">Iris Scissors</td>
			<td>WPI (World Precision Instruments)</td>
			<td>500216-G</td>
			<td>Subcellular fractionation supplies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:433px;">30 mm tissue culture dish</td>
			<td>Fisher Scientific</td>
			<td>08-772B</td>
			<td>Subcellular fractionation supplies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Glass homogenizer and a Teflon pestle</td>
			<td>VWR</td>
			<td>89026-384</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="20" style="height:20px;width:433px;">1.7 mL microcentrifuge tube</td>
			<td>DENVILLE SCIENTIFIC INC.&#160;</td>
			<td>C2170 (1001002)</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="34" style="height:34px;width:433px;">Sorvall Legend XT/XF Centrifuge&#160;</td>
			<td>Thermo Fisher</td>
			<td>75004521</td>
			<td>Subcellular fractionation supplies</td>
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			<td height="22" style="height:22px;width:433px;">Pierce BCA Protein Assay Reagent A, 500 mL</td>
			<td>Thermo Fisher</td>
			<td>#23228</td>
			<td>Western blot supplies</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:433px;">Pierce BCA Protein Assay Reagent B, 25 mL</td>
			<td>Thermo Fisher</td>
			<td>#1859078</td>
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			<td height="21" style="height:21px;width:433px;">SDS-polyacrylamide gel (SDS-PAGE)</td>
			<td>BIO-RAD</td>
			<td>#4561086S</td>
			<td>Western blot supplies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Running Buffer</td>
			<td>Made in the lab</td>
			<td></td>
			<td>Western blot supplies.&#160;</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:433px;">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 height="20">
			<td height="20" style="height:20px;width:433px;">Polyvinyl difluoride (PVDF) membrane&#160;</td>
			<td>Milipore</td>
			<td>IPVH00010</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Transfer Buffer</td>
			<td>Made in the lab</td>
			<td></td>
			<td>Western blot supplies.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycine</td>
			<td>Fisher Scientific</td>
			<td>BP381-5</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium dodecyl sulfate</td>
			<td>SIGMA-ALDRICH</td>
			<td>436143</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A454-4</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>detergent for PSD isolation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Mini Trans-Blot Module&#160;</td>
			<td>BIO-RAD</td>
			<td>#1703935</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Nonfat instant dry milk</td>
			<td>Great value</td>
			<td></td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Multi-purposee rotator&#160;</td>
			<td>Thermo Scientific</td>
			<td>Model-2314</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Hyblot CL Autoradiography Film</td>
			<td>DENVILLE SCIENTIFIC INC.&#160;</td>
			<td>E3018 (1001365)</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Enhanced chemifluorescence substrate&#160;</td>
			<td>Thermo Scientific</td>
			<td>32106</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:433px;">a Konica SRX-101A film processor</td>
			<td>KONICA MINOLTA</td>
			<td>SRX-101A</td>
			<td>Western blot supplies</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Name of Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">PSD-95</td>
			<td>Cell Signaling</td>
			<td>#2507</td>
			<td>Antibody dilution = 1:500-1000, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Rabbit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Synaptophysin</td>
			<td>Cell Signaling</td>
			<td>#4329</td>
			<td>Antibody dilution = 1:500-1000, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Rabbit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">alpha-Tubulin</td>
			<td>Santacruz</td>
			<td>SC-5286</td>
			<td>Antibody dilution = 1:500-1000, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Mouse</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">GluN2B</td>
			<td>Neuromab</td>
			<td>75-097</td>
			<td>Antibody dilution = 1:500-1000, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Mouse</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">GluA2</td>
			<td>Sigma-aldrich</td>
			<td>Sab 4501295</td>
			<td>Antibody dilution = 1:500-1000, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Rabbit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">STEP</td>
			<td>Santacruz</td>
			<td>SC-23892</td>
			<td>Antibody dilution = 1:200-500, time = 9 - 12 h, Reaction Temperature = 4 &#176;C, Host Species = Mouse</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Peroxidas AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoReserch laboratory</td>
			<td>715-035-150</td>
			<td>Antibody dilution = 1:2000-5000, time = 1 h, Reaction Temperature = RT, Host Species = Donkey</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:433px;">Peroxidas AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoReserch laboratory</td>
			<td>711-035-152</td>
			<td>Antibody dilution = 1:2000-5000, 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 electric shock and therefore did not display seizures. A total of 4 sham rats were used. For chronic ECS induction, the rats received one electric shock per day for 7 consecutive days and displayed stage 4-5 tonic-clonic seizures upon each electric shock (Figure 1A-B). A total 8 of rats received chronic ECS induction and a total of 4 sham rats were used as &#34;no seizure&#34; rats. Although most rats that received chronic ECS were behaviorally indistinguishable from sham &#34;no seizure&#34; rats, a few developed body tremors and reduced exploratory activity by the 7th electric shock.
To examine if ECS-induced global elevation of activity alters protein expression in the hippocampi, rats were sacrificed at 3 h and 24 h after acute ECS and at 24 h and 96 h after the final ECS in chronic ECS treatment (Figure 1B). Two hippocampi from each rat were rapidly dissected and subjected to our optimized small-scale fractionation protocol (Figure 2). The initial homogenate of two hippocampi free of insoluble tissue and nuclei (S1 fraction) was re-centrifuged at 13,800 x g for 10 min to separate the supernatant containing soluble cytoplasmic proteins (S2 fraction) from the crude membrane pellet containing synaptosomes (P2 fraction) (Figure 2). Our Western blot detected cytoplasmic protein &#945;-tubulin in the S2 fractions, but not the P2 fractions, of the hippocampi from rats treated with acute and chronic ECS (Figure 3 and Figure 4), demonstrating the successful fractionation of cytosolic soluble proteins from crude membrane pellets.
PSD95 is a member of the membrane-associated guanylate kinase (MAGUK) family and a core component of PSD12,18,25. PSD95 and NMDA receptor subunit GluN2B were detected exclusively in the P2 fractions, but not the S2 fractions (Figure 3 and Figure 4). Stronger expression of another glutamatergic &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2 was detected in the P2 fractions compared to the S2 fractions in the hippocampi (Figure 3 and Figure 4), indicating that postsynaptic membrane proteins are enriched in the crude membrane P2 fraction pellets. Interestingly, synaptic vesicle protein synaptophysin and integral membrane protein Striatal Enriched Tyrosine Phosphatase 61 (STEP61)26 were detected equally in the S2 and P2 fractions (Figure 3 and Figure 4). Considering the small size of synaptic vesicles-with an average diameter of 39 nm27 and endosomes, the centrifugation to isolate the crude membrane pellet (P2 fraction) might not have been sufficient to pellet all synaptic vesicles and endosomes. These results together indicate that our crude fractionation protocol can enrich membrane-bound proteins and transmembrane proteins that are not associated with synaptic vesicles and possibly with endosomes in the crude membrane P2 fraction.
To examine if ECS-induced global elevation of activity alters the expression of postsynaptic proteins in the hippocampi, the crude membrane P2 fraction was lysed in ice-cold water, resuspended in HEPES buffer, and centrifuged at 25,000 x g for 20 min to isolate the lysed pellet (LP1) (Figure 2). The LP1 pellet was resuspended in HEPES buffer containing 1% Triton X-100 detergent and centrifuged at 25,000 x g over 3 h to obtain the PSD pellet (Figure 2). The subsequent Western blot of the PSD fractions detected PSD95, GluN2B, and GluA2 (Figure 3 and Figure 4), which are known postsynaptic proteins17,25,28. However, the PSD fractions lacked &#945;-tubulin and, importantly, presynaptic marker synaptophysin (Figure 3 and Figure 4). STEP61, which dephosphorylates GluN2B and GluA2 and acts as their negative regulator29,30,31, was not found in the PSD fractions (Figure 3 and Figure 4), consistent with the recent report demonstrating the synaptic exclusion of STEP61, mediated by PSD95 enriched in the PSD26. Altogether, these results indicate that our small-scale fractionation method successfully isolates PSD proteins from the crude membrane P2 fraction.
The quantification of Western blotting revealed that GluN2B expression was unaltered in the P2 fraction but displayed an increasing trend in the PSD fraction at 3 h and 24 h after acute ECS (n = 4) (Figure 3B). GluA2 expression was not changed in both the P2 and PSD fractions at 3 h and 24 h following acute ECS (Figure 3C). At 24 h and 96 h after chronic ECS, GluN2B expression displayed a decreasing trend in the P2 fraction and was significantly reduced in the PSD fraction (24 h: p &#60;0.05, n = 4; 48 h: p &#60;0.01, n = 4) (Figure 4B). In contrast, chronic ECS did not affect GluA2 expression in the P2 and PSD fractions (n = 4) (Figure 4C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56016/56016fig1.jpg" /> 
Figure 1: Schema for the Induction of Acute ECS and Chronic ECS. (A) A rat was connected to a pulse generator via ear-clip electrodes and an electrical shock (55 mA, 100 pulses/s for 0.5 s) was applied to elicit stage 4-5 tonic-clonic seizures. (B) Acute ECS was induced by one electric shock. Chronic ECS was elicited by one electric shock per day for 7 consecutive days. The time points shown represent the duration following the induction of acute ECS or the last ECS for chronic ECS induction prior to dissection of the hippocampi. Sham &#34;no seizure&#34; (NS) control rats were handled identically but did not receive an electrical shock. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56016/56016fig2.jpg" /> 
Figure 2: Workflow of Subcellular Fractionation to Isolate the S2, P2, and PSD Fractions from the Hippocampi of a Single Rat. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56016/56016fig3.jpg" /> 
Figure 3: Examination of Synaptic Proteins in Hippocampal S2, P2, and PSD Fractions from the Rats that Received NS or Acute ECS. (A) Representative Western blots show the protein expression of NMDA receptor subunit GluN2B, AMPA receptor GluA2, and STEP61 in the S2, P2, and PSD fractions from the hippocampi of sham &#34;no seizure&#34; (NS) rats and rats that received acute ECS. The cytoplasmic-soluble protein &#945;-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. PSD-95 is enriched in both the P2 and PSD fractions. (B-C) Quantification of GluN2B (B) and GluA2 (C) in the P2 and PSD fractions at 3 and 24 h after acute ECS (n = 4 rats per time point). GluN2B and GluA2 expression in the P2 fraction was normalized to &#945;-Tubulin in the S2 fraction. GluN2B and GluA2 expression in the PSD fraction was normalized to PSD-95 in the PSD fraction. The data are presented as the percent &#177; SEM. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56016/56016fig4.jpg" /> 
Figure 4: Examination of Synaptic Proteins in Hippocampal S2, P2, and PSD Fractions from the Rats that Received NS or Chronic ECS. Representative Western blots of &#945;-tubulin; synaptophysin; STEP61; and postsynaptic proteins, including PSD95, GluN2B, and GluA2, in the S2, P2, and PSD fractions from the hippocampi of sham &#34;no seizure&#34; (NS) rats and rats that received chronic ECS. The cytoplasmic-soluble protein &#945;-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. PSD-95 is enriched in both the P2 and PSD fractions. (B-C) Quantification of GluN2B (B) and GluA2 (C) in the P2 and PSD fractions at 24 and 96 h after chronic ECS (n = 4 rats per time point). GluN2B and GluA2 expression in the P2 fraction was normalized to the &#945;-Tubulin in the S2 fraction. GluN2B and GluA2 expression in the PSD fraction was normalized to PSD-95 in the PSD fraction. The data are presented as percent &#177; SEM; *p &#60;0.05, **p &#60;0.01 compared with control (ANOVA and Tukey&#39;s post-hoc test). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56016/56016fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Fraction</td>
			<td>Protein fraction</td>
			<td>Protein marker</td>
		</tr>
		<tr>
			<td>P1</td>
			<td>Nuclear</td>
			<td>Histon H1</td>
		</tr>
		<tr>
			<td>S1</td>
			<td>Cytosol/membrances&#160;</td>
			<td>a-tubulin (cytoskeleton) and GAPDH (cytosol)</td>
		</tr>
		<tr>
			<td>P2&#160;</td>
			<td>Crude synaptosomes</td>
			<td>AMPA and NMDA receptor subunits</td>
		</tr>
		<tr>
			<td>S2</td>
			<td>Cytosol/light membrances&#160;</td>
			<td>GAPDH (cytosol) and LAMP1 (lysosome)</td>
		</tr>
		<tr>
			<td>Synaptosomal membrane&#160;</td>
			<td>Synaptosome/mitochondria</td>
			<td>AMPA and NMDAR receptor subunits, Synaptophysin (presynaptic marker)</td>
		</tr>
		<tr>
			<td>PSD</td>
			<td>PSD</td>
			<td>AMPA and NMDA receptor subunits, PSD95, PICK1, CaMKII</td>
		</tr>
	</tbody>
</table>
Table 1: List of Protein Markers and Antibodies to Distinguish Subcellular Fractions.

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

Nanyang Technological University

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

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in the late 1960&#8217;s, proteins associated with the synaptic plasma membrane can be isolated by ultracentrifugation on a sucrose density gradient. Once synaptic membranes are isolated, the macromolecular complex known as the post-synaptic density can be subsequently isolated due to its detergent insolubility. The techniques used to isolate synaptic plasma membranes and post-synaptic density proteins remain essentially the same after 40 years, and are widely used in current neuroscience research. This article details the fractionation of proteins associated with the synaptic plasma membrane and post-synaptic density using a discontinuous sucrose gradient. Resulting protein preparations are suitable for western blotting or 2D DIGE analysis.

This article details the enrichment of proteins associated with the synaptic plasma membrane by ultracentrifugation on a discontinuous sucrose gradient. The subsequent preparation of post-synaptic density proteins is also described. Protein preparations are suitable for western blotting or 2D DIGE analysis.

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Non-invasive Assessment of Changes in Corticomotoneuronal Transmission in Humans

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor learning. There exists a number of noninvasive electrophysiological methods investigating the excitability and plasticity of this pathway. However, most methods are based on quantification of compound potentials and neglect that the corticospinal pathway consists of many different connections that are more or less direct. Here, we present a method that allows testing excitability of different fractions of the corticospinal transmission. This so called H-reflex conditioning technique allows one to assess excitability of the fastest (monosynaptic) and also polysynaptic corticospinal pathways. Furthermore, by using two different stimulation sites, the motor cortex and the cervicomedullary junction, it allows not only differentiation between cortical and spinal effects but also assessment of transmission at the corticomotoneural synapse. In this manuscript, we describe how this method can be used to assess corticomotoneural transmission after low-frequency repetitive transcranial magnetic stimulation, a method that was previously shown to reduce excitability of cortical cells. Here we demonstrate that not only cortical cells are affected by this repetitive stimulation but also transmission at the corticomotoneuronal synapse at the spinal level. This finding is important for the understanding of basic mechanisms and sites of neuroplasticity. Besides investigation of basic mechanisms, the H-reflex conditioning technique may be applied to test changes in corticospinal transmission following behavioral (e.g., training) or therapeutic interventions, pathology or aging and therefore allows a better understanding of neural processes that underlie movement control and motor learning.

The aim of the present study was to assess changes in transmission at the corticomotoneuronal synapses in humans after repetitive transcranial magnetic stimulation. For this purpose, an electrophysiological method is introduced that allows assessment of pathway specific corticospinal transmission, i.e. differentiation of fast, direct corticospinal pathways from polysynaptic connections.

The corticospinal pathway is the major pathway connecting the brain with the muscles and is therefore highly relevant for movement control and motor ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Quantifying Acute Changes in Renal Sympathetic Nerve Activity in Response to Central Nervous System Manipulations in Anesthetized Rats

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are limited resources directing scientists in the techniques for measuring and analyzing these variables. This protocol describes the methods for measuring RSNA and mean arterial pressure in anesthetized rats. The protocol also includes the approaches for accessing the brain during RSNA recordings for central nervous system (CNS) manipulations. The RSNA recording technique is compatible with pharmacologic, optogenetic, or electrical stimulation of the CNS. The approach is useful when an investigator will measure short-term (min to h) autonomic responses in non-survival experiments to correlate anatomically with CNS nuclei. The approach is not intended to be used to obtain chronic (survival) recordings of RSNA in rats. Discharges in RSNA, averaged rectified RSNA, and mean arterial pressure can be quantified and analyzed further using parametric statistical tests. Methods for obtaining venous access, recording mean arterial pressure telemetrically, and brain fixation for future histological analysis are also described in the article.

Methods for measuring sympathetic and cardiovascular responses to central nervous system (CNS) manipulations are important for advancing neuroscience. This protocol was developed to assist scientists with measuring and quantifying acute changes in renal sympathetic nerve activity (RSNA) in anesthetized rats (non-survival).

Renal sympathetic nerve activity (RSNA) and mean arterial pressure are important parameters in cardiovascular and autonomic research; however, there are ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...