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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 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.
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 α5 subunit-containing GABAA receptor (α5-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 α5-GABAAR in the prefrontal cortex of mice exposed to UCMS as compared with non-stressed control mice.
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
2. Preparation of the stock solutions
3. Preparation of the workstation
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.
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)
6. Crosslinking reaction
7. Tissue lysis, protein preparation, and western blot
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 gav...
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
The authors report no conflicts of interest.
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.
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 |
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