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 statis...

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha5GABAA+receptor+activity+sets+the+threshold+for+long-term+potentiation+and+constrains+hippocampus-dependent+memory.">Alpha5GABAA receptor activity sets the threshold for long-term potentiation and constrains hippocampus-dependent memory.</a> The Journal of Neuroscience. 30 (15), 5269-5282 (2010).</li><li>Nollet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+depression:+Unpredictable+chronic+mild+stress+in+mice.">Models of depression: Unpredictable chronic mild stress in mice.</a> Current Protocols. 1 (8), e208 (2021).</li><li>Tomoda, T., Sumitomo, A., Newton, D., Sibille, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+origin+of+somatostatin-positive+neuron+vulnerability.">Molecular origin of somatostatin-positive neuron vulnerability.</a> Molecular Psychiatry. 27 (4), 2304-2314 (2022).</li><li>Guilloux, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 (CaCl2)</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&#937;&#183;cm @ 25 &#176;C; total organic carbon (TOC) &#8804; 5 ppb]&#160;</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&#38;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 (MgCl2&#183;6H2O)</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)&#160;</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

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 &#945;5-GABAAR levels in the mouse PFC, we ran 10 &#181;g each of BS3-crosslinked and non-crosslinked protein samples on SDS-PAGE and analyzed the proteins by western blot using an anti-&#945;5-GABAAR antibody (rabbit polyclonal) (Figure 7). The non-crosslinked protein samples gave the total amount of &#945;5-GABAAR at ~55 kDa, while the BS3-crosslinked protein samples gav...

Watch this Scientific Journal Video about BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain at JoVE.com

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

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

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 ...

The chemical crosslinking assay using BS3 as a crosslinker will help in determining the neurotransmitter receptor cell surface level, which is a critical determinant of the efficacy of neuro transmission. This assay is particularly useful for brain region specific evaluation of receptor dynamics in response to various psychotropic agents or psychogenic stress modalities in animal models. To begin, rapidly remove the brain from the skull of the euthanized C57BL/6J mouse.Submerge it in a Petri dish containing ice-cold PBS for 10 to 15 seconds. Place the chilled brain into the brain matrix on ice facing the ventral side up. To cut the brain coronally, insert the first razor blade through the border between the olfactory bulb and the olfactory peduncle.Using three to four additional razor blades, serially cut the anterior part of the brain coronally at one millimeter intervals. Hold the inserted razor blades together to lift the coronal slices off the brain matrix, leaving the posterior part behind. Using forceps, separate the razor blades from one another and place them on the flat, chilled surface with the brain slice facing up.To sample the prefrontal cortex, choose the second and third slices posterior to the first slice containing the olfactory peduncle. Using a tissue punch or forceps, remove the desired region. Put the tissue on the chilled razor blade and evenly divide it into two pieces.Use the fine tip of forceps to mince each tissue into pieces on the razor blade with multiple vertical motions. Spike the pre-chilled labeled tube containing artificial cerebrospinal fluid with 30 microliters of BS3 solution or vehicle solution E.Then immediately transfer the minced tissues into the desired tube. To sample the hippocampus, remove the posterior part of the brain from the matrix and place it on moistened filter paper on a chilled flat surface with the dorsal side facing up.With a curved probe and forceps approaching from the dorsal side, dissect the hippocampus from both hemispheres, then mince the tissue and transfer it into appropriate spiked tubes as previously demonstrated. For crosslinking reaction, invert the tube containing tissue and solution to break the tissue chunks into small pieces. Incubate the samples on the tube rotator at 4 degrees Celsius for 30 minutes to 2 hours and record the start time of the BS3 incubation for each tube.Then quench the reaction with 78 microliters of one molar glycine. Incubate further for 10 minutes at 4 degrees Celsius with constant rotation and record the start and end time for quenching. In the western blot analysis after BS3 crosslinking, the non-crosslinked protein showed the total amount of alpha5-subunit of GABA-A receptor at approximately 55 kilodaltons.However, the BS3 crosslinked protein showed endomembrane-associated alpha5-subunit of GABA-A receptor migrating at approximately 55 kilodaltons, along with higher molecular weight protein species representing protein complexes covalently crosslinked to alpha5-subunit of GABA-A receptor. Moreover, a significant and progressive reduction in surface alpha5-subunit of GABA-A receptor levels was observed in the prefrontal cortex at three weeks and five weeks of UCMS as compared with the no-stress control mice. The receptors are known to shuttle between the plasma membrane surface and internal membrane at ambient temperature.So performing tissue dissection at a low temperature is important. By utilizing crosslinking assay, it was revealed that psychosocial stress can significantly modulate surface levels of GABA receptors which partly serves as a molecular basis for anxiety, behavior, or cognitive deficits.

bs3-chemical-crosslinking-assay-evaluating-effect-chronic-stress-on

Research

JoVE Journal

Neuroscience

945 조회수.  Campbell Family Mental Health Research Institute. BS3를 가교제로 사용하는 화학적 가교 분석은 신경 전달 효능의 중요한 결정 요인인 신경 전달 물질 수용체 세포 표면 수준을 결정하는 데 도움이 됩니다. 이 분석은 동물 모델에서 다양한 향정신성 제제 또는 심인성 스트레스 양식에 반응하는 수용체 역학의 뇌 영역 특이적 평가에 특히 유용합니다. 시작하려면 안락사된 C57BL/6J 마우스의 두개골에서 뇌를 빠르게 제거합니다.