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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The BS3 chemical crosslinking assay reveals reduced cell surface GABAA 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 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.

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 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 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 statis.......

Representative Results

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.......

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

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
0.5 M EDTA, pH 8.0Invitrogen15575020
1 M HEPESGibco15630080
10x TBSBio-Rad1706435
2.5 M (45%, w/v) GlucoseSigmaG8769
2-mercaptoethanolSigmaM3148
4x SDS sample buffer (Laemmli)Bio-Rad1610747
Bis(sulfosuccinimidyl)suberate (BS3)PierceA39266No-Weigh Format; 10 x 2 mg
Brain matrixTed Pella15003For mouse, 30 g adult, coronal, 1 mm
Calcium chloride (CaCl2)SigmaC4901
Curved probeFine Science Tools10088-15Gross Anatomy Probe; angled 45
Deionized watermilli-QEQ 7000Ultrapure water [resistivity 18.2 MΩ·cm @ 25 °C; total organic carbon (TOC) ≤ 5 ppb] 
Dithiothreitol (DTT)Sigma10197777001
Filter paper (3MM)Whatman3030-917
Forceps (large)Fine Science Tools11152-10Extra Fine Graefe Forceps
Forceps (small)Fine Science Tools11251-10Dumont #5 Forceps
GABA-A R alpha 5 antibodyInvitrogenPA5-31163Polyclonal Rabbit IgG; detect erroneous signal upon chemical crosslinking
GABA-A R alpha 5 C-terminus antibodyR&D SystemsPPS027Polyclonal Rabbit IgG; cross-reacts with mouse and rat
GlycineSigmaW328707
Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L)Bio-Rad1721019
Magnesium chloride (MgCl2·6H2O)SigmaM2670
Nonidet-P40, substitute (NP-40)SantaCruz68412-54-4
Potassium chloride (KCl)SigmaP9541
Protease inhibitor cocktailSigmaP8340
PVDF membraneBio-Rad1620177
Scissors (large)Fine Science Tools14007-14Surgical Scissors - Serrated
Scissors (small)Fine Science Tools14060-09Fine Scissors - Sharp
Sodium chloride (NaCl)SigmaS9888
Sonicator (Qsonica Sonicator Q55) Qsonica15338284
Table-top refregerated centrifugeEppendorf5425R
Tissue punch (ID 1 mm)Ted Pella15110-10Miltex Biopsy Punch with Plunger, ID 1.0 mm, OD 1.27 mm
Trans-Blot Turbo 5x Transfer bufferBio-Rad10026938
Tube rotator (LabRoller)LabnetH5000

References

  1. Groc, L., Choquet, D. Linking glutamate receptor movements and synapse function. Science. 368 (6496), (2020).
  2. Diering, G. H., Huganir, R. L. The AMPA receptor code of synaptic plasticity. Neuron. 100 (2....

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