BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA 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 si....

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 numbe....

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 gav.......

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 stress

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