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

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.