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 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).</li>
	<li>Place the animals under UCMS or control non-stressed conditions for 5-8 weeks, following the protocol as described previously14.</li>
	<li>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.</li>
</ol>
2. Preparation of the stock solutions
<ol>
	<li>Prepare and store the following solutions as instructed prior to the assay.
	<ol>
		<li>Prepare 5 M NaCl by dissolving 14.6 g of NaCl in 40 mL of deionized water. Store at room temperature (RT).</li>
		<li>Prepare 1.08 M KCl by dissolving 3.22 g of KCl in 40 mL of deionized water. Store at RT.</li>
		<li>Prepare 400 mM MgCl2 by dissolving 3.25 g of MgCl2&#183;6H2O in 40 mL of deionized water. Store at RT.</li>
		<li>Prepare 1 M glycine by dissolving 3 g of glycine in 40 mL of deionized water, and store at 4 &#176;C.</li>
		<li>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 &#181;m, and aliquot into 2 mL tubes. Store at &#8722;20 &#176;C.</li>
		<li>Prepare 10% Nonidet-P40 (NP-40) by diluting it at a ratio of 1:10 (v/v) in deionized water. Store at RT.</li>
		<li>Prepare 0.5 M EDTA (pH = 8.0), and store at RT.</li>
		<li>Prepare 1 M HEPES buffer (pH = 7.2-7.5), and store at 4 &#176;C.</li>
		<li>Prepare 2.5 M or 45% (w/v) glucose, and store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Preparation of the workstation
<ol>
	<li>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 &#181;L), a pipettor (P200), a stopwatch, a memo pad, a pen, and microcentrifuge tubes (1.5 mL).
	<ol>
		<li>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 &#215; 2 regions &#215; 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 &#181;L, 300 &#181;L] of each sample) (to be used in section 7). Thus, it is required to label 144 tubes in total for this cohort size.</li>
	</ol>
	</li>
	<li>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 &#176;C]), a freezer (&#8722;80 &#176;C) for storing samples, and a bucket of dry ice for the temporary storage of the samples (to be used in section 7)</li>
</ol>
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.
<ol>
	<li>Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 &#181;L 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.</li>
	<li>Prepare the lysis buffer as mentioned in Table 2. Store on ice (400 &#181;L to be used per sample).</li>
	<li>Prepare a 52 mM BS3 stock solution (26x) in 5 mM sodium citrate buffer (pH = 5.0).
	<ol>
		<li>First, prepare 100 mM citric acid (stock A) and 100 mM sodium citrate (stock B).</li>
		<li>Dilute stock A and stock B at a ratio of 1:20 with deionized water. Add 100 &#181;L each to 1.9 mL of water to prepare 5 mM citric acid (stock C) and 5 mM sodium citrate (stock D), respectively.</li>
		<li>Mix 410 &#181;L of stock C and 590 &#181;L of stock D to prepare a 5 mM sodium citrate buffer (pH = 5.0) (solution E, 1 mL).</li>
		<li>Confirm the pH of solution E using a pH indicator strip.</li>
		<li>Dissolve 24 mg of BS3 in 806.4 &#181;L of solution E by vortexing for 30 s to prepare the BS3 stock solution (26x).</li>
		<li>Prepare another 1 mL of solution E to use as vehicle control for the non-crosslinking samples. 
		&#8203;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 &#176;C until use. Once reconstituted, BS3 remains active only for approximately &#8804;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.</li>
	</ol>
	</li>
</ol>
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)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Place the chilled brain into the brain matrix on ice, with the ventral side of the brain facing up (Figure 3).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>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). 
	&#8203;NOTE: The entire dissection time for each animal should be kept at ~5 min for the experimental conditions and results to be consistent.</li>
</ol>
6. Crosslinking reaction
<ol>
	<li>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).</li>
	<li>Spike the tube pre-chilled on ice (from step 4.1) with 30 &#181;L 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.</li>
	<li>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)</li>
	<li>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.</li>
	<li>Quench the reaction by spiking the tube with 78 &#181;L of 1 M glycine, and further incubate the sample for 10 min at 4 &#176;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). 
	&#8203;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.</li>
</ol>
7. Tissue lysis, protein preparation, and western blot
<ol>
	<li>After 10 min of quenching (step 6.5), spin the samples at 20,000 x g at 4 &#176;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.</li>
	<li>Add 400 &#181;L of ice-cold lysis buffer per tube.</li>
	<li>Sonicate the samples for 1 s five times with 5 s intervals in between, while keeping the samples on ice.</li>
	<li>Spin samples at 20,000 x g for 2 min to spin out insoluble tissue debris (pellet), and save the supernatant.</li>
	<li>Use 5 &#181;L of the supernatant for measuring the protein concentration using the bicinchoninic acid (BCA) assay.</li>
	<li>Divide the rest of the supernatant into two tubes; one tube contains 100 &#181;L of supernatant for the subsequent western blot, and the other tube contains the rest (~300 &#181;L) for long-term storage at &#8722;80 &#176;C. Add an appropriate amount of 4x SDS sample buffer (supplemented with &#946;2-mercaptoethanol) to the samples, and incubate at 70 &#176;C for 10 min.</li>
	<li>Run 10-20 &#181;g 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.</li>
	<li>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.</li>
	<li>Briefly wash the membrane twice with TBS-T, and incubate it with the primary antibody diluted in TBS-T overnight at 4 &#176;C.</li>
	<li>Wash off the primary antibody three times for 10 min each in TBS-T at RT.</li>
	<li>Incubate the membrane with horseradish peroxidase-conjugated secondary antibody diluted in TBS-T for 1 h at RT.</li>
	<li>Wash off the secondary antibody three times for 10 min each in TBS-T at RT.</li>
	<li>Incubate the membrane in enhanced chemiluminescence reagent, and detect the signal using the gel-documentation apparatus.</li>
</ol>

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The authors report no conflicts of interest.

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 ...

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Research

JoVE Journal

Neuroscience

1.0K Views.  Campbell Family Mental Health Research Institute. The BS3 chemical crosslinking assay reveals reduced cell surface GABAA receptor expression in mouse brains under chronic psychosocial stress conditions.

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

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

Brain Imaging Investigation of the Impairing Effect of Emotion on Cognition

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional distractibility) effects (reviewed in 1). Complementing our recent protocol 2 describing a method that allows investigation of the neural correlates of the memory-enhancing effect of emotion (see also 1, 3-5), here we present a protocol that allows investigation of the neural correlates of the detrimental impact of emotion on cognition. The main feature of this method is that it allows identification of reciprocal modulations between activity in a ventral neural system, involved in 'hot' emotion processing (HotEmo system), and a dorsal system, involved in higher-level 'cold' cognitive/executive processing (ColdEx system), which are linked to cognitive performance and to individual variations in behavior (reviewed in 1). Since its initial introduction 6, this design has proven particularly versatile and influential in the elucidation of various aspects concerning the neural correlates of the detrimental impact of emotional distraction on cognition, with a focus on working memory (WM), and of coping with such distraction 7,11, in both healthy 8-11 and clinical participants 12-14.

We present a protocol that allows investigation of the neural mechanisms mediating the detrimental impact of emotion on cognition, using functional magnetic resonance imaging. This protocol can be used with both healthy and clinical participants.

Emotions can impact cognition by exerting both enhancing (e.g., better memory for emotional events) and impairing (e.g., increased emotional ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...