Name: BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain (Video) | JoVE Journal
Uploaded: 2023-05-26T12:15:00.000+00:00
Duration: 5 min 17 s
Description: Learn the BS3 chemical crosslinking assay to evaluate GABA A receptor surface expression in mouse brain tissue. This method reveals how chronic psychosocial stress alters receptor dynamics in the prefrontal cortex.

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The authors thank the CAMH animal facility staff for caring for the animals over the study duration. This work was supported by the Canadian Institute of Health Research (CIHR Project Grant #470458 to T.T.), the Discovery Fund from the CAMH (to T.P.), the National Alliance for Research on Schizophrenia and Depression (NARSAD award #25637 to E.S.), and the Campbell Family Mental Health Research Institute (to E.S.). E.S. is the founder of Damona Pharmaceuticals, a biopharma dedicated to bringing novel GABAergic compounds to the clinic.

All the animal work in this protocol was completed in accordance with the Ontario Animals for Research Act (RSO 1990, Chapter A.22) and the Canadian Council on Animal Care (CCAC) and was approved by the Institutional Animal Care Committee.
1. Preparation of animals
<ol>
	<li>Determine the animal numbers to be used in the experiments, and divide them into appropriate groups or experimental cohorts. See the discussion section for a discussion of the group size, sex, and statistical power. 
	NOTE: This protocol is customized for mice (C57BL6/J strain; 2-4 months of age; typically 20-30 g of body weight; equivalent numbers of males and females to be used).</li>
	<li>Place the animals under UCMS or control non-stressed conditions for 5-8 weeks, following the protocol as described previously14.</li>
	<li>After the last UCMS procedure, allow the animals to stay in their home cages for 1 day before using them for the crosslinking assay to avoid acute stress effects on receptor expression.</li>
</ol>
2. Preparation of the stock solutions
<ol>
	<li>Prepare and store the following solutions as instructed prior to the assay.
	<ol>
		<li>Prepare 5 M NaCl by dissolving 14.6 g of NaCl in 40 mL of deionized water. Store at room temperature (RT).</li>
		<li>Prepare 1.08 M KCl by dissolving 3.22 g of KCl in 40 mL of deionized water. Store at RT.</li>
		<li>Prepare 400 mM MgCl2 by dissolving 3.25 g of MgCl2&#183;6H2O in 40 mL of deionized water. Store at RT.</li>
		<li>Prepare 1 M glycine by dissolving 3 g of glycine in 40 mL of deionized water, and store at 4 &#176;C.</li>
		<li>Prepare 1 M dithiothreitol (DTT) by dissolving 1.54 g of DTT in 10 mL of deionized water. Filter-sterilize it through a filter with a pore size of 0.2 &#181;m, and aliquot into 2 mL tubes. Store at &#8722;20 &#176;C.</li>
		<li>Prepare 10% Nonidet-P40 (NP-40) by diluting it at a ratio of 1:10 (v/v) in deionized water. Store at RT.</li>
		<li>Prepare 0.5 M EDTA (pH = 8.0), and store at RT.</li>
		<li>Prepare 1 M HEPES buffer (pH = 7.2-7.5), and store at 4 &#176;C.</li>
		<li>Prepare 2.5 M or 45% (w/v) glucose, and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Preparation of the workstation
<ol>
	<li>On the day of the BS3 crosslinking assay, gather the following materials in the animal dissection room (Figure 1), with several items pre-chilled on ice: an ice bucket, a metal temperature block (pre-chilled on ice), ice-cold PBS in a 50 mL conical tube and frozen PBS in a Petri dish, filter paper moistened with PBS and placed on a chilled flat surface or blue ice, a brain matrix (1 mm interval for razor blade insertion) pre-chilled on ice, razor blades (~10) pre-chilled on ice, dissection tools (scissors, forceps, a curved probe), a tissue punch, 70% ethanol spray wiping paper and paper towels, pipet tips (200 &#181;L), a pipettor (P200), a stopwatch, a memo pad, a pen, and microcentrifuge tubes (1.5 mL).
	<ol>
		<li>Prior to the assay, label the microcentrifuge tubes with the sample information (e.g., animal ID number, treatment type [UCMS versus no stress (NS)], brain region, with or without BS3, etc.). 
		NOTE: For the BS3 crosslinking assays, two samples from each brain region must be collected; one sample will be used for crosslinking (with BS3) and the other for the non-crosslinking reaction (without BS3) as a control. Therefore, in order to sample from two brain regions (i.e., the prefrontal cortex [PFC] and hippocampus [HPC]) in 12 mice, label 48 tubes for initial sampling (= 2 samples &#215; 2 regions &#215; 12 mice) (to be used in section 6). Label an additional two sets of 48 tubes for later storage (for storing two different volumes [100 &#181;L, 300 &#181;L] of each sample) (to be used in section 7). Thus, it is required to label 144 tubes in total for this cohort size.</li>
	</ol>
	</li>
	<li>In addition, make sure the following equipment is available in the laboratory: a table-top refrigerated microcentrifuge, a sonicator, a tube rotator (to be used in the cold room or inside the refrigerator [4 &#176;C]), a freezer (&#8722;80 &#176;C) for storing samples, and a bucket of dry ice for the temporary storage of the samples (to be used in section 7)</li>
</ol>
4. Preparation of the working solutions and buffers
NOTE: On the morning of the assay, prepare the following solutions. This calculation is based on the necessary solutions to process two brain regions (i.e., the PFC and HPC) from 12 mice.
<ol>
	<li>Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 &#181;L of aCSF into each sampling tube (the 48 tubes labeled in step 3.1.1), and place them in the metal temperature block on ice to pre-chill the buffer.</li>
	<li>Prepare the lysis buffer as mentioned in Table 2. Store on ice (400 &#181;L to be used per sample).</li>
	<li>Prepare a 52 mM BS3 stock solution (26x) in 5 mM sodium citrate buffer (pH = 5.0).
	<ol>
		<li>First, prepare 100 mM citric acid (stock A) and 100 mM sodium citrate (stock B).</li>
		<li>Dilute stock A and stock B at a ratio of 1:20 with deionized water. Add 100 &#181;L each to 1.9 mL of water to prepare 5 mM citric acid (stock C) and 5 mM sodium citrate (stock D), respectively.</li>
		<li>Mix 410 &#181;L of stock C and 590 &#181;L of stock D to prepare a 5 mM sodium citrate buffer (pH = 5.0) (solution E, 1 mL).</li>
		<li>Confirm the pH of solution E using a pH indicator strip.</li>
		<li>Dissolve 24 mg of BS3 in 806.4 &#181;L of solution E by vortexing for 30 s to prepare the BS3 stock solution (26x).</li>
		<li>Prepare another 1 mL of solution E to use as vehicle control for the non-crosslinking samples. 
		&#8203;NOTE: Prepare BS3 stock solution when everything else is ready and the experiment is about to begin. The BS3 should be stored desiccated at 4 &#176;C until use. Once reconstituted, BS3 remains active only for approximately &#8804;3 h. As the pH of the 5 mM sodium citrate buffer (solution E) is reported to rise over time, causing accelerated BS3 hydrolysis, it is recommended that solution E is prepared fresh from stock solutions A and B9. Due to the limited solubility of BS3 at low temperatures, keep the reconstituted BS3 at RT. Use up the reconstituted BS3 in 3 h, and do not freeze/thaw or reuse the reconstituted BS3.</li>
	</ol>
	</li>
</ol>
5. Dissection of brain tissues
NOTE: From this step on, at least two people should work together in a coordinated manner. While one person focuses on the animal dissection (steps 5.2-5.10 and step 6.3), the other person should work as a timekeeper and help coordinate the assay (step 5.1, step 6.1, step 6.2, step 6.4, and step 6.5)
<ol>
	<li>Bring the first animal for dissection from the housing area to the dissection room. 
	NOTE: As acute stressors (e.g., a novel environment, the smell of blood) may affect the brain protein dynamics, the animals should be kept in their home cages placed far from the dissection area and then be brought individually into the dissection room for immediate decapitation.</li>
	<li>Euthanize the mouse by cervical dislocation followed by decapitation. Remove the brain rapidly out of the skull, and submerge it in ice-cold PBS in a Petri dish for 10-15 s (Figure 2). 
	NOTE: The animals are not anesthetized for BS3 experiments since any anesthetic agent could potentially influence the surface presentation level of the neurotransmitter receptors9.</li>
	<li>Place the chilled brain into the brain matrix on ice, with the ventral side of the brain facing up (Figure 3).</li>
	<li>Insert the first razor blade through the border between the olfactory bulb and the olfactory peduncle to cut the brain coronally (Figure 4). Using three to four additional razor blades, serially cut the anterior part of the brain coronally with 1 mm intervals.</li>
	<li>Lift the coronal slices off the brain matrix by holding all the inserted razor blades together, leaving the posterior part of the brain behind in the brain matrix. Use forceps to separate the razor blades from one another, and place them on the flat, chilled surface with the brain slice facing up (Figure 5).</li>
	<li>Identify the slices containing the region of interest. For sampling the PFC, choose the second and third slices posterior to the first slice containing the olfactory peduncle.</li>
	<li>Remove the region of interest using a tissue punch (Video 1), put it aside on the chilled razor blade, and evenly divide the tissue into two (e.g., tissues from the left versus right hemisphere, with one half to be used for the BS3 crosslinking reaction and the other half for the no-crosslinking control if the target protein of interest is equally expressed in both hemispheres).</li>
	<li>Mince each tissue into pieces on the razor blade using the fine tip of forceps with multiple vertical motions against the blade (Video 2) instead of mashing or grinding the tissues. This will maximize the surface area accessible to BS3 without severely compromising the membrane integrity of the cells. Immediately after mincing, transfer the minced tissues into the appropriate tubes (see step 6.3).</li>
	<li>For sampling the HPC, take the posterior part of the brain out of the matrix, and place the brain on moistened filter paper on the chilled flat surface, with the dorsal side facing up (Figure 6).</li>
	<li>Using a curved probe and forceps, and approaching from the dorsal side (Video 3), dissect out the HPC (dorsal half, ventral half, or both) from both hemispheres (one half for BS3 crosslinking and the other half for the control). Mince each tissue as in step 5.8, and transfer the tissue into appropriate tubes (see step 6.3). 
	&#8203;NOTE: The entire dissection time for each animal should be kept at ~5 min for the experimental conditions and results to be consistent.</li>
</ol>
6. Crosslinking reaction
<ol>
	<li>Bring the next animal for dissection from the housing area to the dissection room when the dissection of the brain of the previous mouse is about to finish (step 5.10).</li>
	<li>Spike the tube pre-chilled on ice (from step 4.1) with 30 &#181;L of BS3 solution (26x) or vehicle solution E right before the minced tissues are ready to be transferred into the appropriate tubes. Change the pipet tips in between tubes to ensure no BS3 is contaminated in the no-crosslinking control tubes.</li>
	<li>Transfer the minced tissues (from step 5.8 and step 5.10) into the appropriate tubes, and then start dissecting the next animal (step 5.2)</li>
	<li>Invert and mix the tube to break the tissue chunks apart into smaller pieces (Video 4), and start incubating the samples on the tube rotator in the cold room for 30 min to 2 h. Record the start time of the BS3 incubation for each tube. See the discussion section for the optimal incubation time.</li>
	<li>Quench the reaction by spiking the tube with 78 &#181;L of 1 M glycine, and further incubate the sample for 10 min at 4 &#176;C with constant rotation. Record the start and end time of quenching for each tube. Continue assisting the person focusing on the animal dissection by bringing the next animal (step 6.1) and helping with crosslinking (step 6.2). 
	&#8203;NOTE: Treat each sample with the exact same timing across all the samples. If the dissection room is far away from the cold room, it is highly recommended that a third person be recruited to take part in the sample incubation and quenching in the cold room.</li>
</ol>
7. Tissue lysis, protein preparation, and western blot
<ol>
	<li>After 10 min of quenching (step 6.5), spin the samples at 20,000 x g at 4 &#176;C for 2 min, and discard the supernatant. If a third person is available, proceed to step 7.2; otherwise, snap-freeze the samples on dry ice, and pause the assay here until the brain dissection (section 5) and crosslinking (section 6) for all the animals are completed.</li>
	<li>Add 400 &#181;L of ice-cold lysis buffer per tube.</li>
	<li>Sonicate the samples for 1 s five times with 5 s intervals in between, while keeping the samples on ice.</li>
	<li>Spin samples at 20,000 x g for 2 min to spin out insoluble tissue debris (pellet), and save the supernatant.</li>
	<li>Use 5 &#181;L of the supernatant for measuring the protein concentration using the bicinchoninic acid (BCA) assay.</li>
	<li>Divide the rest of the supernatant into two tubes; one tube contains 100 &#181;L of supernatant for the subsequent western blot, and the other tube contains the rest (~300 &#181;L) for long-term storage at &#8722;80 &#176;C. Add an appropriate amount of 4x SDS sample buffer (supplemented with &#946;2-mercaptoethanol) to the samples, and incubate at 70 &#176;C for 10 min.</li>
	<li>Run 10-20 &#181;g of protein per well on an acrylamide gel for electrophoresis (SDS-PAGE), and then transfer proteins to the polyvinylidene fluoride (PVDF) membrane for western blot analysis.</li>
	<li>Block the PVDF membrane in 5% (w/v) skim milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at RT.</li>
	<li>Briefly wash the membrane twice with TBS-T, and incubate it with the primary antibody diluted in TBS-T overnight at 4 &#176;C.</li>
	<li>Wash off the primary antibody three times for 10 min each in TBS-T at RT.</li>
	<li>Incubate the membrane with horseradish peroxidase-conjugated secondary antibody diluted in TBS-T for 1 h at RT.</li>
	<li>Wash off the secondary antibody three times for 10 min each in TBS-T at RT.</li>
	<li>Incubate the membrane in enhanced chemiluminescence reagent, and detect the signal using the gel-documentation apparatus.</li>
</ol>

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The authors report no conflicts of interest.

Although the impact of chronic psychosocial stress on behaviors (i.e., emotionality and cognitive deficits) and molecular changes (i.e., reduced expression of GABAergic genes and accompanying deficits in GABAergic neurotransmission) are well-documented10, the mechanisms underlying such deficits need further investigation. In particular, given the recent study showing that chronic stress significantly affects the neuronal proteome through overload on the ER functions and, thus, elevated ER stress<s...

Neurotransmitter receptors are localized either at the neuronal plasma membrane surface or intracellularly on the endomembranes (e.g., the endosome, the endoplasmic reticulum [ER], or the trans-Golgi apparatus) and dynamically shuttle between these two compartments depending on intrinsic physiological states in neurons or in response to extrinsic neural network activities1,2. Since newly secreted neurotransmitters elicit their physiological functions primarily through the surface-localized pool of receptors, the surface receptor levels for a given neurotransmitter are one of the critical determinants of its signaling capacity within the neural circuit3. 
Several methods are available to monitor surface receptor levels in cultured neurons, including the surface biotinylation assay4, the immunofluorescence assay with a specific antibody in non-permeabilized conditions5, or the use of a receptor transgene genetically fused with a pH-sensitive fluorescent optical indicator (e.g., pHluorin)6. By contrast, these approaches are either limited or impractical when assessing surface receptor levels in vivo. For example, the surface biotinylation procedure may not be practical for processing large quantities and sample numbers of in vivo brain tissues due to its relatively high price and the subsequent steps necessary for purifying the biotinylated proteins on avidin-conjugated beads. For neurons embedded in three-dimensional brain architecture, low antibody accessibility or difficulties in microscope-based quantification may pose a significant limitation for assessing the surface receptor levels in vivo. To visualize the distribution of neurotransmitter receptors in intact brains, non-invasive methods, such as positron emission tomography, could be used to measure receptor occupancy and estimate the surface receptor levels7. However, this approach critically relies on the availability of specific radio ligands, expensive equipment, and special expertise, making it less accessible for routine use by most researchers.
Here, we describe a simple, versatile method for measuring surface receptor levels in experimental animal brains ex vivo using a water-soluble, membrane-impermeable chemical crosslinker, bis(sulfosuccinimidyl)suberate (BS3)8,9. BS3 targets primary amines in the side chain of lysine residues and can covalently crosslink proteins in close vicinity to each other. When brain slices are freshly prepared from a region of interest and incubated in a buffer containing BS3, the cell surface receptors are crosslinked with neighboring proteins and, thus, transform into higher-molecular weight species, whereas the intracellular endomembrane-associated receptors remain unmodified. Therefore, the surface and intracellular receptor pools can be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitated by western blot using antibodies specific to the receptor to be studied. 
Unpredictable chronic mild stress (UCMS) is a well-established experimental paradigm for inducing chronic psychosocial stress in rodents10. UCMS elicits anxiety-/depressive-like behavioral phenotypes and cognitive deficits via the modulation of an array of neurotransmitter systems, including GABA and its receptors10,11. In particular, the &#945;5 subunit-containing GABAA receptor (&#945;5-GABAAR) is implicated in the regulation of memory and cognitive functions12,13, suggesting the possible involvement of altered functions of this subunit in UCMS-induced cognitive deficits. In this protocol, we used the BS3 crosslinking assay to quantitate levels of surface-expressed &#945;5-GABAAR in the prefrontal cortex of mice exposed to UCMS as compared with non-stressed control mice.

<table><tbody><tr><td>0.5 M EDTA, pH 8.0</td><td>Invitrogen</td><td>15575020</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>10x TBS</td><td>Bio-Rad</td><td>1706435</td><td></td></tr><tr><td>2.5 M (45%, w/v) Glucose</td><td>Sigma</td><td>G8769</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M3148</td><td></td></tr><tr><td>4x SDS sample buffer (Laemmli)</td><td>Bio-Rad</td><td>1610747</td><td></td></tr><tr><td>Bis(sulfosuccinimidyl)suberate (BS3)</td><td>Pierce</td><td>A39266</td><td>No-Weigh Format; 10 x 2 mg</td></tr><tr><td>Brain matrix</td><td>Ted Pella</td><td>15003</td><td>For mouse, 30 g adult, coronal, 1 mm</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>Curved probe</td><td>Fine Science Tools</td><td>10088-15</td><td>Gross Anatomy Probe; angled 45</td></tr><tr><td>Deionized water</td><td>milli-Q</td><td>EQ 7000</td><td>Ultrapure water [resistivity 18.2 M&amp;Omega;&amp;middot;cm @ 25 &amp;deg;C; total organic carbon (TOC) &amp;le; 5 ppb]&amp;nbsp;</td></tr><tr><td>Dithiothreitol (DTT)</td><td>Sigma</td><td>10197777001</td><td></td></tr><tr><td>Filter paper (3MM)</td><td>Whatman</td><td>3030-917</td><td></td></tr><tr><td>Forceps (large)</td><td>Fine Science Tools</td><td>11152-10</td><td>Extra Fine Graefe Forceps</td></tr><tr><td>Forceps (small)</td><td>Fine Science Tools</td><td>11251-10</td><td>Dumont #5 Forceps</td></tr><tr><td>GABA-A R alpha 5 antibody</td><td>Invitrogen</td><td>PA5-31163</td><td>Polyclonal Rabbit IgG; detect erroneous signal upon chemical crosslinking</td></tr><tr><td>GABA-A R alpha 5 C-terminus antibody</td><td>R&amp;amp;D Systems</td><td>PPS027</td><td>Polyclonal Rabbit IgG; cross-reacts with mouse and rat</td></tr><tr><td>Glycine</td><td>Sigma</td><td>W328707</td><td></td></tr><tr><td>Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L)</td><td>Bio-Rad</td><td>1721019</td><td></td></tr><tr><td>Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>Nonidet-P40, substitute (NP-40)</td><td>SantaCruz</td><td>68412-54-4</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>PVDF membrane</td><td>Bio-Rad</td><td>1620177</td><td></td></tr><tr><td>Scissors (large)</td><td>Fine Science Tools</td><td>14007-14</td><td>Surgical Scissors - Serrated</td></tr><tr><td>Scissors (small)</td><td>Fine Science Tools</td><td>14060-09</td><td>Fine Scissors - Sharp</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>Sonicator (Qsonica Sonicator Q55)&amp;nbsp;</td><td>Qsonica</td><td>15338284</td><td></td></tr><tr><td>Table-top refregerated centrifuge</td><td>Eppendorf</td><td>5425R</td><td></td></tr><tr><td>Tissue punch (ID 1 mm)</td><td>Ted Pella</td><td>15110-10</td><td>Miltex Biopsy Punch with Plunger, ID 1.0 mm, OD 1.27 mm</td></tr><tr><td>Trans-Blot Turbo 5x Transfer buffer</td><td>Bio-Rad</td><td>10026938</td><td></td></tr><tr><td>Tube rotator (LabRoller)</td><td>Labnet</td><td>H5000</td><td></td></tr></tbody></table>

bs3 chemical crosslinking assay: evaluating the effect of chronic stress on cell surface gabaa receptor presentation in the rodent brain

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the animal kingdom and can be recognized as either adaptive or maladaptive responses to a wide range of stress modalities. Rodents provide a proven experimental model for translational studies addressing the integrative mechanisms of anxiety at the molecular, cellular, and circuit levels. In particular, the chronic psychosocial stress paradigm elicits maladaptive responses mimicking anxiety-/depressive-like behavioral phenotypes that are analogous between humans and rodents. While previous studies show significant effects of chronic stress on neurotransmitter contents in the brain, the effect of stress on neurotransmitter receptor levels is understudied. In this article, we present an experimental method to quantitate the neuronal surface levels of neurotransmitter receptors in mice under chronic stress, especially focusing on gamma-aminobutyric acid (GABA) receptors, which are implicated in the regulation of emotion and cognition. Using the membrane-impermeable irreversible chemical crosslinker, bissulfosuccinimidyl suberate (BS3), we show that chronic stress significantly downregulates the surface availability of GABAA receptors in the prefrontal cortex. The neuronal surface levels of GABAA receptors are the rate-limiting process for GABA neurotransmission and could, therefore, be used as a molecular marker or a proxy of the degree of anxiety-/depressive-like phenotypes in experimental animal models. This crosslinking approach is applicable to a variety of receptor systems for neurotransmitters or neuromodulators expressed in any brain region and is expected to contribute to a deeper understanding of the mechanisms underlying emotion and cognition.

To demonstrate the feasibility of the BS3 crosslinking assay for evaluating the surface α5-GABAAR levels in the mouse PFC, we ran 10 µg each of BS3-crosslinked and non-crosslinked protein samples on SDS-PAGE and analyzed the proteins by western blot using an anti-α5-GABAAR antibody (rabbit polyclonal) (Figure 7). The non-crosslinked protein samples gave the total amount of α5-GABAAR at ~55 kDa, while the BS3-crosslinked protein samples gave a certain amount o...

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...

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Research

JoVE Journal

Neuroscience

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.

Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

Stress granules (SGs) are cytoplasmic RNA granules containing stalled ribonucleoprotein particles (RNPs), and important in cellular response to various stresses. Dynamics of SGs can be followed in live cells by visualizing the localization of a tagged component of SGs in transfected primary cells after stress.

Visualization of G3BP Stress Granules Dynamics in Live Primary Cells

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

By integrating four daily social stressors, the present study describes a multiple integrated social stress (MISS) model for psychiatric disorders in female C57BL/6J mice. After ten days of repeated MISS exposure, the mice developed depressive- and anxiety-like phenotypes, thus providing a natural, etiology-based animal model for studying psychiatric disorders in female subjects.

A Multiple Integrated Social Stress Model for Psychiatric Disorders in Female C57BL/6J Mice

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Restraint to Induce Stress in Mice and Rats

Learn the BS3 chemical crosslinking assay to evaluate GABA A receptor surface expression in mouse brain tissue. This method reveals how chronic psychosocial stress alters receptor dynamics in the prefrontal cortex.

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain (Video) | JoVE Journal

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABA_A Receptor Presentation in the Rodent Brain

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