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>

<ol>
	<li>Groc, L., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+glutamate+receptor+movements+and+synapse+function.">Linking glutamate receptor movements and synapse function.</a> Science. 368 (6496), (2020).</li><li>Diering, G. H., Huganir, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+AMPA+receptor+code+of+synaptic+plasticity.">The AMPA receptor code of synaptic plasticity.</a> Neuron. 100 (2), 314-329 (2018).</li><li>Tomoda, T., Hikida, T., Sakurai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+DISC1+in+neuronal+trafficking+and+its+implication+in+neuropsychiatric+manifestation+and+neurotherapeutics.">Role of DISC1 in neuronal trafficking and its implication in neuropsychiatric manifestation and neurotherapeutics.</a> Neurotherapeutics. 14 (3), 623-629 (2017).</li><li>Sumitomo, A., 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=Ulk2+controls+cortical+excitatory-inhibitory+balance+via+autophagic+regulation+of+p62+and+GABAA+receptor+trafficking+in+pyramidal+neurons.">Ulk2 controls cortical excitatory-inhibitory balance via autophagic regulation of p62 and GABAA receptor trafficking in pyramidal neurons.</a> Human Molecular Genetics. 27 (18), 3165-3176 (2018).</li><li>Brady, M. L., Jacob, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+localization+of+&#945;5+GABA+(A)+receptors+via+gephyrin+interaction+regulates+dendritic+outgrowth+and+spine+maturation.">Synaptic localization of &#945;5 GABA (A) receptors via gephyrin interaction regulates dendritic outgrowth and spine maturation.</a> Developmental Neurobiology. 75 (11), 1241-1251 (2015).</li><li>Jacob, T. C., 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=Gephyrin+regulates+the+cell+surface+dynamics+of+synaptic+GABAA+receptors.">Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.</a> The Journal of Neuroscience. 25 (45), 10469-10478 (2005).</li><li>Takamura, Y., Kakuta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+receptor+visualization+and+evaluation+of+receptor+occupancy+with+positron+emission+tomography.">In vivo receptor visualization and evaluation of receptor occupancy with positron emission tomography.</a> Journal of Medicinal Chemistry. 64 (9), 5226-5251 (2021).</li><li>Archibald, K., Perry, M. J., Moln&#225;r, E., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+expression+and+metabolic+half-life+of+AMPA+receptors+in+cultured+rat+cerebellar+granule+cells.">Surface expression and metabolic half-life of AMPA receptors in cultured rat cerebellar granule cells.</a> Neuropharmacology. 37 (10-11), 1345-1353 (1998).</li><li>Boudreau, A. C., 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=A+protein+crosslinking+assay+for+measuring+cell+surface+expression+of+glutamate+receptor+subunits+in+the+rodent+brain+after+in+vivo+treatments.">A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments.</a> Current Protocols in Neuroscience. , 1-19 (2012).</li><li>Fee, C., Banasr, M., 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=Somatostatin-positive+gamma-aminobutyric+acid+interneuron+deficits+in+depression:+Cortical+microcircuit+and+therapeutic+perspectives.">Somatostatin-positive gamma-aminobutyric acid interneuron deficits in depression: Cortical microcircuit and therapeutic perspectives.</a> Biological Psychiatry. 82 (8), 549-559 (2017).</li><li>Bernardo, A., 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=Symptomatic+and+neurotrophic+effects+of+GABAA+receptor+positive+allosteric+modulation+in+a+mouse+model+of+chronic+stress.">Symptomatic and neurotrophic effects of GABAA receptor positive allosteric modulation in a mouse model of chronic stress.</a> Neuropsychopharmacology. 47 (9), 1608-1619 (2022).</li><li>Pr&#233;vot, T., 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=Altered+GABA-mediated+information+processing+and+cognitive+dysfunctions+in+depression+and+other+brain+disorders.">Altered GABA-mediated information processing and cognitive dysfunctions in depression and other brain disorders.</a> Molecular Psychiatry. 26 (1), 151-167 (2021).</li><li>Martin, L. 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. P., 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=Molecular+evidence+for+BDNF-+and+GABA-related+dysfunctions+in+the+amygdala+of+female+subjects+with+major+depression.">Molecular evidence for BDNF- and GABA-related dysfunctions in the amygdala of female subjects with major depression.</a> Molecular Psychiatry. 17 (11), 1130-1142 (2012).</li><li>Lin, L. C., 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=Somatostatin,+neuronal+vulnerability+and+behavioral+emotionality.">Somatostatin, neuronal vulnerability and behavioral emotionality.</a> Molecular Psychiatry. 20 (3), 377-387 (2015).</li><li>Fritschy, J. M., Mohler, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAA-receptor+heterogeneity+in+the+adult+rat+brain:+differential+regional+and+cellular+distribution+of+seven+major+subunits.">GABAA-receptor heterogeneity in the adult rat brain: differential regional and cellular distribution of seven major subunits.</a> The Journal of Comparative Neurology. 359 (1), 154-194 (1995).</li><li>Rubio, F. J., Li, X., Liu, Q. R., Cimbro, R., Hope, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+activated+cell+sorting+(FACS)+and+gene+expression+analysis+of+Fos-expressing+neurons+from+fresh+and+frozen+rat+brain+tissue.">Fluorescence activated cell sorting (FACS) and gene expression analysis of Fos-expressing neurons from fresh and frozen rat brain tissue.</a> Journal of Visualized Experiments. (114), e54358 (2016).</li><li>Boudreau, A. C., Wolf, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+sensitization+to+cocaine+is+associated+with+increased+AMPA+receptor+surface+expression+in+the+nucleus+accumbens.">Behavioral sensitization to cocaine is associated with increased AMPA receptor surface expression in the nucleus accumbens.</a> The Journal of Neuroscience. 25 (40), 9144-9151 (2005).</li><li>Conrad, K. L., 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=Formation+of+accumbens+GluR2-lacking+AMPA+receptors+mediates+incubation+of+cocaine+craving.">Formation of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving.</a> Nature. 454 (7200), 118-121 (2008).</li><li>Tomoda, T., 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=BDNF+controls+GABAAR+trafficking+and+related+cognitive+processes+via+autophagic+regulation+of+p62.">BDNF controls GABAAR trafficking and related cognitive processes via autophagic regulation of p62.</a> Neuropsychopharmacology. 47 (2), 553-563 (2022).</li><li>Hernandez-Rabaza, V., 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=Sildenafil+reduces+neuroinflammation+and+restores+spatial+learning+in+rats+with+hepatic+encephalopathy:+Underlying+mechanisms.">Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: Underlying mechanisms.</a> Journal of Neuroinflammation. 12, 195 (2015).</li></ol>

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><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 (CaCl&lt;sub&gt;2&lt;/sub&gt;)</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&amp;Omega;&amp;middot;cm @ 25 &amp;deg;C; total organic carbon (TOC) &amp;le; 5 ppb]&amp;nbsp;</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&amp;amp;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 (MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;6H&lt;sub&gt;2&lt;/sub&gt;O)</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)&amp;nbsp;</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...

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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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JoVE Journal

Neuroscience

1.1K 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

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Behavior

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Medicine

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Biochemistry

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

A Chronic Immobilization Stress Protocol for Inducing Depression-Like Behavior in Mice

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, therapeutic treatments for depression or research on depression is scarce. Thus, in the present paper, we propose a mouse model of depression induced by movement restriction. Chronic mild stress (CMS) is a well-known technique to induce depressive-like behavior. However, it necessitates a complex procedure consisting of a combination of various mild stresses. In contrast, chronic immobilization stress (CIS) is a readily accessible chronic stress model, modified from a restraint model that induces depressive behavior by restricting movement using a restrainer for a certain period. To evaluate the depressive-like behaviors, the sucrose preference test (SPT), the tail suspension test (TST), and the ELISA assay to measure stress marker corticosterone levels are combined in the present experiment. The described protocols illustrate the induction of CIS and evaluation of the changes in behavior and physiological factors for the validation of depression.

This article provides a simplified and standardized protocol for induction of depressive-like behavior in chronically immobilized mice by using a restrainer. In addition, behavior and physiological techniques to verify induction of depression are explained.

Depression is not yet fully understood, but various causative factors have been reported. Recently, the prevalence of depression has increased. However, ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

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

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