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

Summary

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

Abstract

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 GABA_A receptors in the prefrontal cortex. The neuronal surface levels of GABA_A 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.

Introduction

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 activities^1,2. 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 receptors^10,11. In particular, the α5 subunit-containing GABA_A receptor (α5-GABA_AR) is implicated in the regulation of memory and cognitive functions^12,13, 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 α5-GABA_AR in the prefrontal cortex of mice exposed to UCMS as compared with non-stressed control mice.

Protocol

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

1. 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).
2. Place the animals under UCMS or control non-stressed conditions for 5-8 weeks, following the protocol as described previously¹⁴.
3. 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.

2. Preparation of the stock solutions

1. Prepare and store the following solutions as instructed prior to the assay.
   1. Prepare 5 M NaCl by dissolving 14.6 g of NaCl in 40 mL of deionized water. Store at room temperature (RT).
   2. Prepare 1.08 M KCl by dissolving 3.22 g of KCl in 40 mL of deionized water. Store at RT.
   3. Prepare 400 mM MgCl₂ by dissolving 3.25 g of MgCl₂·6H₂O in 40 mL of deionized water. Store at RT.
   4. Prepare 1 M glycine by dissolving 3 g of glycine in 40 mL of deionized water, and store at 4 °C.
   5. 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 µm, and aliquot into 2 mL tubes. Store at −20 °C.
   6. Prepare 10% Nonidet-P40 (NP-40) by diluting it at a ratio of 1:10 (v/v) in deionized water. Store at RT.
   7. Prepare 0.5 M EDTA (pH = 8.0), and store at RT.
   8. Prepare 1 M HEPES buffer (pH = 7.2-7.5), and store at 4 °C.
   9. Prepare 2.5 M or 45% (w/v) glucose, and store at 4 °C.

3. Preparation of the workstation

1. 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 µL), a pipettor (P200), a stopwatch, a memo pad, a pen, and microcentrifuge tubes (1.5 mL).
   1. 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 × 2 regions × 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 µL, 300 µL] of each sample) (to be used in section 7). Thus, it is required to label 144 tubes in total for this cohort size.
2. 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 °C]), a freezer (−80 °C) for storing samples, and a bucket of dry ice for the temporary storage of the samples (to be used in section 7)

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.

1. Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 µL 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.
2. Prepare the lysis buffer as mentioned in Table 2. Store on ice (400 µL to be used per sample).
3. Prepare a 52 mM BS3 stock solution (26x) in 5 mM sodium citrate buffer (pH = 5.0).
   1. First, prepare 100 mM citric acid (stock A) and 100 mM sodium citrate (stock B).
   2. Dilute stock A and stock B at a ratio of 1:20 with deionized water. Add 100 µL each to 1.9 mL of water to prepare 5 mM citric acid (stock C) and 5 mM sodium citrate (stock D), respectively.
   3. Mix 410 µL of stock C and 590 µL of stock D to prepare a 5 mM sodium citrate buffer (pH = 5.0) (solution E, 1 mL).
   4. Confirm the pH of solution E using a pH indicator strip.
   5. Dissolve 24 mg of BS3 in 806.4 µL of solution E by vortexing for 30 s to prepare the BS3 stock solution (26x).
   6. Prepare another 1 mL of solution E to use as vehicle control for the non-crosslinking samples.
      ​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 °C until use. Once reconstituted, BS3 remains active only for approximately ≤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.

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)

1. 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.
2. 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.
3. Place the chilled brain into the brain matrix on ice, with the ventral side of the brain facing up (Figure 3).
4. 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.
5. 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).
6. 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.
7. 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).
8. 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).
9. 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).
10. 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).
    ​NOTE: The entire dissection time for each animal should be kept at ~5 min for the experimental conditions and results to be consistent.

6. Crosslinking reaction

1. 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).
2. Spike the tube pre-chilled on ice (from step 4.1) with 30 µL 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.
3. 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)
4. 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.
5. Quench the reaction by spiking the tube with 78 µL of 1 M glycine, and further incubate the sample for 10 min at 4 °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).
   ​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.

7. Tissue lysis, protein preparation, and western blot

1. After 10 min of quenching (step 6.5), spin the samples at 20,000 x g at 4 °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.
2. Add 400 µL of ice-cold lysis buffer per tube.
3. Sonicate the samples for 1 s five times with 5 s intervals in between, while keeping the samples on ice.
4. Spin samples at 20,000 x g for 2 min to spin out insoluble tissue debris (pellet), and save the supernatant.
5. Use 5 µL of the supernatant for measuring the protein concentration using the bicinchoninic acid (BCA) assay.
6. Divide the rest of the supernatant into two tubes; one tube contains 100 µL of supernatant for the subsequent western blot, and the other tube contains the rest (~300 µL) for long-term storage at −80 °C. Add an appropriate amount of 4x SDS sample buffer (supplemented with β2-mercaptoethanol) to the samples, and incubate at 70 °C for 10 min.
7. Run 10-20 µg 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.
8. 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.
9. Briefly wash the membrane twice with TBS-T, and incubate it with the primary antibody diluted in TBS-T overnight at 4 °C.
10. Wash off the primary antibody three times for 10 min each in TBS-T at RT.
11. Incubate the membrane with horseradish peroxidase-conjugated secondary antibody diluted in TBS-T for 1 h at RT.
12. Wash off the secondary antibody three times for 10 min each in TBS-T at RT.
13. Incubate the membrane in enhanced chemiluminescence reagent, and detect the signal using the gel-documentation apparatus.

Representative Results

To demonstrate the feasibility of the BS3 crosslinking assay for evaluating the surface α5-GABA_AR 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-GABA_AR antibody (rabbit polyclonal) (Figure 7). The non-crosslinked protein samples gave the total amount of α5-GABA_AR at ~55 kDa, while the BS3-crosslinked protein samples gave a certain amount of endomembrane-associated α5-GABA_AR (migrating at ~55 kDa) along with higher-molecular weight protein species representing protein complexes covalently crosslinked to α5-GABA_AR. For the quantification of the surface α5-GABA_AR levels, we can assess the extent of depletion of α5-GABA_AR at ~55 kDa from the total pool upon crosslinking, as the subunit then shifts to higher-molecular weight positions. Practically, the surface levels of α5-GABA_AR can be calculated by subtracting the amount of endomembrane-associated α5-GABA_AR (at ~55 kDa in the crosslinked sample lane) from the total α5-GABA_AR levels (at ~55 kDa in the non-crosslinked sample lane).

We next evaluated the effects of UCMS (at 3 weeks and 5 weeks) on the surface and total α5-GABA_AR levels in the mouse PFC. In this experiment, in order to follow the time course of the crosslinking reaction, we prepared the samples at 1 h, 2 h, and 3 h after the addition of BS3. Since BS3 crosslinking reactions appeared to reach a plateau by 2 h, the data at the 1 h time point were used to plot the graph. We observed a significant and progressive reduction in surface α5-GABA_AR levels in the PFC at 3 weeks and 5 weeks of UCMS, as compared with the no-stress control mice (Figure 8). The data showed negligible or no apparent changes in the total receptor levels under these experimental conditions, suggesting that chronic stress specifically impacted α5-GABA_AR trafficking to the cell surface.

Figure 1: Dissection tools used in the BS3 crosslinking assay. Filter paper is moistened with ice-cold PBS and placed on the flat surface of blue ice. A Petri dish filled with PBS and stored at −20 °C 1 day prior to the assay is placed on ice. The brain matrix and razor blades are pre-chilled on ice. Scissors (one large, one small), forceps (one large, a few small ones), and a tissue punch are all cleaned prior to the assay. Please click here to view a larger version of this figure.

Figure 2: Whole mouse brain dissected out of the skull and placed in ice-cold PBS. Immediately after the whole brain was removed from the skull, it was submerged in ice-cold PBS for 10-15 s in a Petri dish on ice. This slows down brain metabolism and minimizes the protein trafficking and degradation while also helping the tissue to become harder, which makes the subsequent brain slicing easier. Please click here to view a larger version of this figure.

Figure 3: A mouse brain placed in the brain matrix. The chilled mouse brain submerged in ice-cold PBS was transferred to the brain matrix and placed with the ventral side facing up. The razor blades and the matrix were pre-chilled on ice. Please click here to view a larger version of this figure.

Figure 4: Coronal sectioning of a mouse brain in the brain matrix. The first pre-chilled razor blade was inserted through the border between the olfactory bulb and the olfactory peduncle to start sectioning the brain coronally. Please click here to view a larger version of this figure.

Figure 5: Serial coronal sections of a mouse brain. The anterior part of the mouse brain was cut coronally by inserting five razor blades serially into the brain matrix (1 mm intervals). All the inserted blades were held together, lifted off the matrix, separated from one another using forceps, and placed on the chilled flat surface with the brain slice facing up. (Top left) The olfactory bulb; (middle left) the first section; the second (bottom left) and third (top right) sections were used for sampling the PFC tissues; (bottom right) the fourth section. Please click here to view a larger version of this figure.

Figure 6: Posterior part of the brain for dissecting the hippocampus. After the anterior part of the brain was coronally cut with razor blades (left), the posterior part of the brain (middle) was taken out of the brain matrix and placed on PBS-moistened filter paper on the chilled surface (right). Please click here to view a larger version of this figure.

Figure 7: BS3 crosslinking of GABA_AR on the cell surface. In the presence of BS3, α5-GABA_AR on the plasma membrane surface is crosslinked (XL) with anonymous proteins close to it, such as other GABA_AR subunits within the pentameric GABA_AR assembly or additional neighboring proteins (X), but not with proteins (Y) far away from it. Thus, the α5-GABA_AR appears as a high-molecular weight (HMW) protein species on the blot. α5-GABA_AR on the endosome remains intact and migrates at the expected size of ~55 kDa. Please click here to view a larger version of this figure.

Figure 8: Surface α5-GABA_AR levels affected by UCMS. Both the total and surface levels of α5-GABA_AR in the PFC of mice under the no-stress and UCMS (3 weeks and 5 weeks) conditions were evaluated. To follow the time course of the crosslinking reaction, the samples were prepared at 1 h, 2 h, and 3 h after the addition of BS3. Female mice (2-3 months, N = 4/group) were used. The intact α5-GABA_AR levels at 55 kDa for each condition were first normalized by the corresponding αTubulin levels and then used to calculate the total and endomembrane-associated α5-GABA_AR levels (from the no-crosslinked [No Xlink] and crosslinked [Xlink] samples, respectively). Subsequently, the surface receptor levels were calculated by subtracting the endomembrane-associated amount from the total levels. Since the BS3 crosslinking reactions appeared to reach a plateau by 2 h, the data at the 1 h time point were used to plot the graph (mean ± SEM). Significant effects of chronic stress on surface α5-GABA_AR levels were observed, and this effect was UCMS duration-dependent, as a progressive decrease in the surface α5-GABA_AR levels was identified across the UCMS period. *p < 0.05, **p < 0.01 (Kruskal-Wallis test with Dunn's multiple comparisons). Please click here to view a larger version of this figure.

Working conc.	Stock solution	Amount of stock solution to dispense
1.2 mM CaCl2	480 mM (400x)*	100 μL
20 mM HEPES	1 M (50x)	800 μL
147 mM NaCl	5 M (34x)	1176.5 μL
2.7 mM KCl	1.08 M (400x)	100 μL
1 mM MgCl2	400 mM (400x)	100 μL
10 mM Glucose	2.5 M (250x)	160 μL
Deionized water		37.563 mL
Total		40 mL
* CaCl2 stock should be freshly prepared on the day of experiment.

Table 1: The composition of artificial cerebrospinal fluid.

Working conc.	Stock solution	Amount of stock solution to dispense
25 mM HEPES	1 M (40x)	500 μL
500 mM NaCl	5 M (10x)	2 mL
2 mM EDTA	0.5 M (250x)	80 μL
1 mM DTT	1 M (1000x)	20 μL
0.1% NP-40	10% (100x)	200 μL
Protease inhibitor cocktail	100x	200 μL
Deionized water		17 mL
Total		20 mL

Table 2: The composition of the lysis buffer

Video 1: Isolating the PFC using a tissue punch. Please click here to download this Video.

Video 2: Mincing the PFC using forceps. Please click here to download this Video.

Video 3: Dissecting the HPC using a curved probe and forceps. Please click here to download this Video.

Video 4: Inversion-mixing a tissue sample. Please click here to download this Video.

Discussion

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 stress¹⁵, a question remains as to whether chronic stress affects GABA_AR trafficking through the ER membrane and how altered trafficking or surface levels of GABA_AR could be causally linked to psychopathology.

The BS3 crosslinking assay shown in this protocol will serve as a powerful approach for answering some of these questions. For example, UCMS is known to elicit a series of behavioral changes, including cognitive deficits, anxiety-like behavior, and anhedonic phenotypes, depending on the duration of UCMS¹⁰. Therefore, it would be possible to study the time course and the degree of UCMS-induced changes in surface α5-GABA_AR levels by sampling mouse brains at successive time points from the start of UCMS (e.g., 1 week, 3 weeks, 5 weeks), and it would also be possible to cross-compare with the behavioral phenotypes observed at each time point. Additional GABA_AR subtypes (e.g., α1, α2) and the receptor for neuromodulators known to be affected by chronic stress (e.g., the TrkB receptor for brain-derived neurotrophic factor [BDNF])¹⁰ could be tested simultaneously using the same samples. Since some of these receptor systems and subtypes are selectively implicated in specific behavioral domains (e.g., sedation, anxiety, cognition)¹¹, it is worth correlating the behavioral changes with the type and degree of receptors affected by UCMS at each time point.

Below are the critical points to consider for several steps in the protocol. The first step is to determine the necessary and sufficient numbers of animals to use based on the experimental design and power analysis. For example, to study the effect of chronic stress on surface GABA_A receptor levels, we routinely prepare one group of mice (N = 6/sex) to be exposed to UCMS for 5 weeks and another group (N = 6/sex) to be kept under no-stress conditions. This group size (N = 24 in total, including both sexes) is expected to give enough statistical power to detect a ~20% difference in receptor levels, thus allowing evaluation of both stress effects and sex effects. Notably, it is reported that chronic stress causes sex-dependent differences in behavioral and molecular outcomes¹⁰. For example, women are generally more prone to developing depressive symptoms than men. Consistently, our studies using human postmortem and rodent brains indicate higher levels of behavioral and molecular pathologies in female subjects; downregulated levels of somatostatin (SST), a molecular marker for depression, are more robust in women among depressed patients¹⁶, and increased emotionality is more robust in female mouse models replicating aspects of depressive pathology than in males^15,17. Therefore, it is advised that any experimental design addressing the effects of chronic stress on psychopathological outcomes should include adequate numbers of both males and females to ensure statistical power in analyzing the data and to identify possible sex-dependent effects.

The optimal incubation time with BS3 should be determined in pilot experiments for each receptor to be studied. It is reported that receptor trafficking may occur slowly, even at low temperatures during sample incubation⁹. To capture accurate surface receptor levels at the time of brain dissection, it would be ideal to minimize the incubation time and choose the time point right before the crosslinking reaction reaches the plateau (30 min to 2 h at 4 °C).

We noticed several operational limitations associated with BS3 crosslinking assays. (1) First, due to the relatively short half-life (2-3 h) of BS3 caused by spontaneous hydrolysis within the physiological pH range, one needs to complete the animal dissection and crosslinking reaction within this time frame. This led us to limit the number of animals we could dissect at a time to a maximum of 12. If the experimenter plans on dissecting more than 12 animals, it is advised to divide the experimental cohort into several groups, with each containing less than 12 animals. After the assay for one group is completed, a new batch of BS3 should be freshly prepared to use in subsequent crosslinking assays for the next group. In the same vein, the number of regions of interest to be dissected from one animal should be limited. We routinely sample from two brain regions (PFC, HPC) for the crosslinking assay, and this is the maximum number of brain regions we can dissect in order to obtain consistent results with minimal variability across samples. (2) Second, the specificity of the antibodies used in western blot should be carefully evaluated. The BS3 chemical crosslinking reaction may have an unexpected effect on the antigenicity of the proteins detected by each antibody. We found that one α5-GABA_AR antibody (specified in the Table of Materials) erroneously detected a strong ~50 kDa band specifically in BS3-crosslinked samples, regardless of the presence or absence of the α5-GABA_AR protein in the sample; this ~50 kDa band was seen even in tissue samples from α5-GABA_AR knockout mice, suggesting that this antibody began cross-reacting with irrelevant antigens accidentally generated by the BS3 crosslinking reaction. It is advised that the antibody specificity be thoroughly determined with and without BS3 crosslinking reactions, ideally using knockout tissue samples, if available, for a given protein of interest. (3) The current protocol described here does not address the cell types in which each GABA_AR subtype is expressed (e.g., neurons and astrocytes). For some GABA_AR subtypes (e.g., α1, α5) that are predominantly expressed in neurons¹⁸, the crosslinking assay data obtained using bulk brain tissues, as described in this protocol, should provide information reflecting the neuronal surface levels. However, for other GABA_AR subtypes (e.g., α2) that are highly expressed in both neurons and astrocytes¹⁸, it is of interest to study the receptor surface levels in neurons versus astrocytes separately. To this end, conventional cell sorting (e.g., fluorescence-activated cell sorting [FACS]¹⁹) may be integrated into the BS3 crosslinking protocol; the cell dissociation step for cell sorting can be done after the BS3 quenching step, but one needs to validate that all the procedures and conditions for FACS (e.g., the buffers to use, temperature, incubation time) are compatible with those in the crosslinking assay. In addition, the FACS approach may be used only for GABA_AR subtypes known to be localized to the perisomatic cellular compartment (e.g., α2) but not for the subtypes enriched in distal dendrites (e.g., α5)¹⁸, because peripheral or distal cell compartments are likely lost during the extensive cell dissociation step necessary for cell sorting. (4) Finally, we found the results to be more consistent when we calculated the surface receptor levels by subtracting the amount of endomembrane-associated receptors from the total amount of receptors rather than directly evaluating the surface levels based on the densitometry of high-molecular weight protein species. This is likely because the transfer efficiency of these high-molecular weight protein species onto the PVDF membrane is more variable than that of the original, intact protein of a smaller size (e.g., ~55 kDa for α5-GABA_AR). It is, therefore, recommended to follow the method described in the results section and the legend of Figure 8 to calculate the surface receptor levels.

Apart from the effects of chronic stress on GABA_AR, the BS3 crosslinking assay can also be applied to genetically modified mice or rodent models with experimental manipulations to investigate a number of neurological or neuropsychiatric conditions. This assay has been successfully used to capture cocaine-induced effects on the surface expression of glutamate receptors in the nucleus accumbens of rat brains^20,21. The assay has also been used to show reduced surface expression of α5-GABA_AR in the PFC of heterozygous BDNF knockout mice²². In another previous study, in the model of hepatic encephalopathy in rats, the accompanying spatial learning deficits were causally linked to altered surface expression of glutamate and GABA_A receptors based on this crosslinking assay²³. In summary, the BS3 chemical crosslinking assay provides a versatile tool for capturing brain region-specific and context-dependent changes in a range of receptor systems in the brain, as well as in virtually any other peripheral tissues or organs. This assay can also be conducted in parallel with other methods for evaluating the surface receptor levels, such as the electrophysiological recording of tonic inhibition (especially in the case of α5-GABA_AR), cryogenic electron microscopy, and surface biotinylation, to cross-compare and validate the results.

Materials

Name	Company	Catalog Number	Comments
0.5 M EDTA, pH 8.0	Invitrogen	15575020
1 M HEPES	Gibco	15630080
10x TBS	Bio-Rad	1706435
2.5 M (45%, w/v) Glucose	Sigma	G8769
2-mercaptoethanol	Sigma	M3148
4x SDS sample buffer (Laemmli)	Bio-Rad	1610747
Bis(sulfosuccinimidyl)suberate (BS3)	Pierce	A39266	No-Weigh Format; 10 x 2 mg
Brain matrix	Ted Pella	15003	For mouse, 30 g adult, coronal, 1 mm
Calcium chloride (CaCl2)	Sigma	C4901
Curved probe	Fine Science Tools	10088-15	Gross Anatomy Probe; angled 45
Deionized water	milli-Q	EQ 7000	Ultrapure water [resistivity 18.2 MΩ·cm @ 25 °C; total organic carbon (TOC) ≤ 5 ppb]
Dithiothreitol (DTT)	Sigma	10197777001
Filter paper (3MM)	Whatman	3030-917
Forceps (large)	Fine Science Tools	11152-10	Extra Fine Graefe Forceps
Forceps (small)	Fine Science Tools	11251-10	Dumont #5 Forceps
GABA-A R alpha 5 antibody	Invitrogen	PA5-31163	Polyclonal Rabbit IgG; detect erroneous signal upon chemical crosslinking
GABA-A R alpha 5 C-terminus antibody	R&D Systems	PPS027	Polyclonal Rabbit IgG; cross-reacts with mouse and rat
Glycine	Sigma	W328707
Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L)	Bio-Rad	1721019
Magnesium chloride (MgCl2·6H2O)	Sigma	M2670
Nonidet-P40, substitute (NP-40)	SantaCruz	68412-54-4
Potassium chloride (KCl)	Sigma	P9541
Protease inhibitor cocktail	Sigma	P8340
PVDF membrane	Bio-Rad	1620177
Scissors (large)	Fine Science Tools	14007-14	Surgical Scissors - Serrated
Scissors (small)	Fine Science Tools	14060-09	Fine Scissors - Sharp
Sodium chloride (NaCl)	Sigma	S9888
Sonicator (Qsonica Sonicator Q55)	Qsonica	15338284
Table-top refregerated centrifuge	Eppendorf	5425R
Tissue punch (ID 1 mm)	Ted Pella	15110-10	Miltex Biopsy Punch with Plunger, ID 1.0 mm, OD 1.27 mm
Trans-Blot Turbo 5x Transfer buffer	Bio-Rad	10026938
Tube rotator (LabRoller)	Labnet	H5000