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

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 numbers of males and females to be used).
2. Place the animals under UCMS or control non-stressed conditions for 5-8 weeks, following the protocol as described previously¹⁴.
3. 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.

2. Preparation of the stock solutions

1. Prepare and store the following solutions as instructed prior to the assay.
   1. Prepare 5 M NaCl by dissolving 14.6 g of NaCl in 40 mL of deionized water. Store at room temperature (RT).
   2. Prepare 1.08 M KCl by dissolving 3.22 g of KCl in 40 mL of deionized water. Store at RT.
   3. Prepare 400 mM MgCl₂ by dissolving 3.25 g of MgCl₂·6H₂O in 40 mL of deionized water. Store at RT.
   4. Prepare 1 M glycine by dissolving 3 g of glycine in 40 mL of deionized water, and store at 4 °C.
   5. 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 µm, and aliquot into 2 mL tubes. Store at −20 °C.
   6. Prepare 10% Nonidet-P40 (NP-40) by diluting it at a ratio of 1:10 (v/v) in deionized water. Store at RT.
   7. Prepare 0.5 M EDTA (pH = 8.0), and store at RT.
   8. Prepare 1 M HEPES buffer (pH = 7.2-7.5), and store at 4 °C.
   9. Prepare 2.5 M or 45% (w/v) glucose, and store at 4 °C.

3. Preparation of the workstation

1. 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 µL), a pipettor (P200), a stopwatch, a memo pad, a pen, and microcentrifuge tubes (1.5 mL).
   1. 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 × 2 regions × 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 µL, 300 µL] of each sample) (to be used in section 7). Thus, it is required to label 144 tubes in total for this cohort size.
2. 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 °C]), a freezer (−80 °C) for storing samples, and a bucket of dry ice for the temporary storage of the samples (to be used in section 7)

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.

1. Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 µL 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.
2. Prepare the lysis buffer as mentioned in Table 2. Store on ice (400 µL to be used per sample).
3. Prepare a 52 mM BS3 stock solution (26x) in 5 mM sodium citrate buffer (pH = 5.0).
   1. First, prepare 100 mM citric acid (stock A) and 100 mM sodium citrate (stock B).
   2. Dilute stock A and stock B at a ratio of 1:20 with deionized water. Add 100 µL each to 1.9 mL of water to prepare 5 mM citric acid (stock C) and 5 mM sodium citrate (stock D), respectively.
   3. Mix 410 µL of stock C and 590 µL of stock D to prepare a 5 mM sodium citrate buffer (pH = 5.0) (solution E, 1 mL).
   4. Confirm the pH of solution E using a pH indicator strip.
   5. Dissolve 24 mg of BS3 in 806.4 µL of solution E by vortexing for 30 s to prepare the BS3 stock solution (26x).
   6. Prepare another 1 mL of solution E to use as vehicle control for the non-crosslinking samples.
      ​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 °C until use. Once reconstituted, BS3 remains active only for approximately ≤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.

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)

1. 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.
2. 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.
3. Place the chilled brain into the brain matrix on ice, with the ventral side of the brain facing up (Figure 3).
4. 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.
5. 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).
6. 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.
7. 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).
8. 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).
9. 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).
10. 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).
    ​NOTE: The entire dissection time for each animal should be kept at ~5 min for the experimental conditions and results to be consistent.

6. Crosslinking reaction

1. 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).
2. Spike the tube pre-chilled on ice (from step 4.1) with 30 µL 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.
3. 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)
4. 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.
5. Quench the reaction by spiking the tube with 78 µL of 1 M glycine, and further incubate the sample for 10 min at 4 °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).
   ​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.

7. Tissue lysis, protein preparation, and western blot

1. After 10 min of quenching (step 6.5), spin the samples at 20,000 x g at 4 °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.
2. Add 400 µL of ice-cold lysis buffer per tube.
3. Sonicate the samples for 1 s five times with 5 s intervals in between, while keeping the samples on ice.
4. Spin samples at 20,000 x g for 2 min to spin out insoluble tissue debris (pellet), and save the supernatant.
5. Use 5 µL of the supernatant for measuring the protein concentration using the bicinchoninic acid (BCA) assay.
6. Divide the rest of the supernatant into two tubes; one tube contains 100 µL of supernatant for the subsequent western blot, and the other tube contains the rest (~300 µL) for long-term storage at −80 °C. Add an appropriate amount of 4x SDS sample buffer (supplemented with β2-mercaptoethanol) to the samples, and incubate at 70 °C for 10 min.
7. Run 10-20 µg 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.
8. 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.
9. Briefly wash the membrane twice with TBS-T, and incubate it with the primary antibody diluted in TBS-T overnight at 4 °C.
10. Wash off the primary antibody three times for 10 min each in TBS-T at RT.
11. Incubate the membrane with horseradish peroxidase-conjugated secondary antibody diluted in TBS-T for 1 h at RT.
12. Wash off the secondary antibody three times for 10 min each in TBS-T at RT.
13. Incubate the membrane in enhanced chemiluminescence reagent, and detect the signal using the gel-documentation apparatus.