Name: a rat methyl-seq platform to identify epigenetic changes associated with stress exposure
Uploaded: 2018-10-24T14:45:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure at JoVE.com

This study was funded by NIH grant MH101392 (RSL) and support from the following awards and foundations: a NARSAD Young Investigator Award, Margaret Ann Price Investigator Fund, the James Wah Mood Disorders Scholar Fund via the Charles T. Bauer Foundation, Baker Foundation, and the Project Match Foundation (RSL).

<ol>
	<li>Barski, 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=High-resolution+profiling+of+histone+methylations+in+the+human+genome.">High-resolution profiling of histone methylations in the human genome.</a> Cell. 129 (4), 823-837 (2007).</li><li>Meissner, 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=Genome-scale+DNA+methylation+maps+of+pluripotent+and+differentiated+cells.">Genome-scale DNA methylation maps of pluripotent and differentiated cells.</a> Nature. 454 (7205), 766-770 (2008).</li><li>Bibikova, M., 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=High+density+DNA+methylation+array+with+single+CpG+site+resolution.">High density DNA methylation array with single CpG site resolution.</a> Genomics. 98 (4), 288-295 (2011).</li><li>Naumov, V. 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=Genome-scale+analysis+of+DNA+methylation+in+colorectal+cancer+using+Infinium+HumanMethylation450+BeadChips.">Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips.</a> Epigenetics. 8 (9), 921-934 (2013).</li><li>Wockner, L. F., 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=Genome-wide+DNA+methylation+analysis+of+human+brain+tissue+from+schizophrenia+patients.">Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients.</a> Translational Psychiatry. 4, e339 (2014).</li><li>Meissner, 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=Reduced+representation+bisulfite+sequencing+for+comparative+high-resolution+DNA+methylation+analysis.">Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis.</a> Nucleic Acids Research. 33 (18), 5868-5877 (2005).</li><li>Smith, Z. D., Gu, H., Bock, C., Gnirke, A., Meissner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bisulfite+sequencing+in+mammalian+genomes.">High-throughput bisulfite sequencing in mammalian genomes.</a> Methods. 48 (3), 226-232 (2009).</li><li>Slieker, R. 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=Identification+and+systematic+annotation+of+tissue-specific+differentially+methylated+regions+using+the+Illumina+450k+array.">Identification and systematic annotation of tissue-specific differentially methylated regions using the Illumina 450k array.</a> Epigenetics Chromatin. 6 (1), 26 (2013).</li><li>Hing, B., 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=Adaptation+of+the+targeted+capture+Methyl-Seq+platform+for+the+mouse+genome+identifies+novel+tissue-specific+DNA+methylation+patterns+of+genes+involved+in+neurodevelopment.">Adaptation of the targeted capture Methyl-Seq platform for the mouse genome identifies novel tissue-specific DNA methylation patterns of genes involved in neurodevelopment.</a> Epigenetics. 10 (7), 581-596 (2015).</li><li>Seifuddin, F., 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=Genome-wide+Methyl-Seq+analysis+of+blood-brain+targets+of+glucocorticoid+exposure.">Genome-wide Methyl-Seq analysis of blood-brain targets of glucocorticoid exposure.</a> Epigenetics. 12 (8), 637-652 (2017).</li><li>Irizarry, R. 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=The+human+colon+cancer+methylome+shows+similar+hypo-+and+hypermethylation+at+conserved+tissue-specific+CpG+island+shores.">The human colon cancer methylome shows similar hypo- and hypermethylation at conserved tissue-specific CpG island shores.</a> Nature Genetics. 41 (2), 178-186 (2009).</li><li>Lee, R. S., 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=Adaptation+of+the+CHARM+DNA+methylation+platform+for+the+rat+genome+reveals+novel+brain+region-specific+differences.">Adaptation of the CHARM DNA methylation platform for the rat genome reveals novel brain region-specific differences.</a> Epigenetics. 6 (11), 1378-1390 (2011).</li><li>Jankord, R., 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=Stress+vulnerability+during+adolescent+development+in+rats.">Stress vulnerability during adolescent development in rats.</a> Endocrinology. 152 (2), 629-638 (2011).</li><li>Pellow, S., Chopin, P., File, S. E., Briley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+open:closed+arm+entries+in+an+elevated+plus-maze+as+a+measure+of+anxiety+in+the+rat.">Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat.</a> Journal of Neuroscience Methods. 14 (3), 149-167 (1985).</li><li>Krueger, F., Andrews, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bismark:+a+flexible+aligner+and+methylation+caller+for+Bisulfite-Seq+applications.">Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications.</a> Bioinformatics. 27 (11), 1571-1572 (2011).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biology. 10 (3), R25 (2009).</li><li>Hansen, K. D., Langmead, B., Irizarry, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BSmooth:+from+whole+genome+bisulfite+sequencing+reads+to+differentially+methylated+regions.">BSmooth: from whole genome bisulfite sequencing reads to differentially methylated regions.</a> Genome Biology. 13 (10), R83 (2012).</li><li>Lee, R. S., 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=Chronic+corticosterone+exposure+increases+expression+and+decreases+deoxyribonucleic+acid+methylation+of+Fkbp5+in+mice.">Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice.</a> Endocrinology. 151 (9), 4332-4343 (2010).</li><li>Lee, R. S., 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+measure+of+glucocorticoid+load+provided+by+DNA+methylation+of+Fkbp5+in+mice.">A measure of glucocorticoid load provided by DNA methylation of Fkbp5 in mice.</a> Psychopharmacology. , (2011).</li><li>Bose, M., Olivan, B., Laferrere, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+and+obesity:+the+role+of+the+hypothalamic-pituitary-adrenal+axis+in+metabolic+disease.">Stress and obesity: the role of the hypothalamic-pituitary-adrenal axis in metabolic disease.</a> Current Opinion in Endocrinology, Diabetes, and Obesity. 16 (5), 340-346 (2009).</li><li>Brydon, L., Magid, K., Steptoe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelets,+coronary+heart+disease,+and+stress.">Platelets, coronary heart disease, and stress.</a> Brain, Behavior, and Immunity. 20 (2), 113-119 (2006).</li><li>McKlveen, J. M., 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=Chronic+Stress+Increases+Prefrontal+Inhibition:+A+Mechanism+for+Stress-Induced+Prefrontal+Dysfunction.">Chronic Stress Increases Prefrontal Inhibition: A Mechanism for Stress-Induced Prefrontal Dysfunction.</a> Biological Psychiatry. 80 (10), 754-764 (2016).</li><li>Akalin, 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=methylKit:+a+comprehensive+R+package+for+the+analysis+of+genome-wide+DNA+methylation+profiles.">methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles.</a> Genome Biology. 13 (10), R87 (2012).</li><li>Cervera-Juanes, R., Wilhelm, L. J., Park, B., Grant, K. A., Ferguson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcohol-dose-dependent+DNA+methylation+and+expression+in+the+nucleus+accumbens+identifies+coordinated+regulation+of+synaptic+genes.">Alcohol-dose-dependent DNA methylation and expression in the nucleus accumbens identifies coordinated regulation of synaptic genes.</a> Translational Psychiatry. 7 (1), e994 (2017).</li><li>Cervera-Juanes, R., Wilhelm, L. J., Park, B., Grant, K. A., Ferguson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+the+nucleus+accumbens+identifies+DNA+methylation+signals+differentiating+low/binge+from+heavy+alcohol+drinking.">Genome-wide analysis of the nucleus accumbens identifies DNA methylation signals differentiating low/binge from heavy alcohol drinking.</a> Alcohol. 60, 103-113 (2017).</li></ol>

The manuscript is part of a contest prize from Agilent Technologies.

In this study, we designed and implemented the Methyl-Seq platform for the rat genome. By demonstrating its utility with a rat model of stress, we demonstrated that the experimental and analytical pipeline can provide differentially methylated regions between two comparison groups.
To ensure a successful implementation of the platform, several critical steps need to be observed. First, initial DNA quality and quantity has a significant impact on the quality and quantity of the final Methyl-Seq library. We used a fluorometer, rather than a spectrophotometer, to ensure that our DNA measurement reflected the quantity of double-stranded DNA present. The bioanalyzer was used to measure the molecular size and quantity of DNA following shearing and after adapter ligation. Verifying the molecular size &#34;shift&#34; between these steps is crucial to confirm the presence of adapters at the ends of each DNA fragment that will undergo adapter-mediated PCR in the subsequent steps. The quantity of DNA remaining at the end of the adapter ligation step is also important, since at least 100 ng of the library product is needed at this step to ensure sufficient quantity is available after the target enrichment and bisulfite conversion steps. A final high-sensitivity measurement was performed on the constructed Methyl-Seq library so that the library can be properly diluted for subsequent clustering on the next-generation sequencer. Finally, bisulfite pyrosequencing was employed as a highly-quantitative, independent method to assess the accuracy of the analytical pipeline. The final validation using the original samples and replication using additional animals are crucial steps to ensure that the experiment can detect biologically significant changes in DNA methylation.
We also include several guidelines in the event of deviation from the protocol or if problems are encountered. First, it is possible to lose too much DNA during end-repair, adapter ligation, or magnetic bead purification steps. Alternatively, starting amounts of DNA could be small (&#60;200 ng) due to limited tissue/DNA availability or implementation of various enrichment methods such as fluorescence activated cell sorting. Increasing the cycle number during the two library amplification steps may be able to compensate for the excessive loss of DNA or low starting DNA amount throughout the library construction protocol. However, no more than an additional 2&#8211;3 cycles are recommended, as excessive template amplification is likely to lead to an increase in the number of duplicate reads being sequenced. These duplicates are excluded during the alignment step to prevent bias in percent methylation calculations. Second, if the average DNA size does not increase by more than 30 bps, check to ensure that the reagents are new, as T4 DNA polymerase, Klenow, and/or T4 ligase may be old. Commercially available replacement reagents can be used.
Additionally, it is possible that the predicted DMRs might not validate by pyrosequencing, where DNA methylation differences do not exist or are significantly less than those predicted by analysis. Poor validation of candidate regions is a problem too common for many genome-wide analyses, such as when pyrosequencing results do not confirm differential methylation or the effect size is much smaller than that predicted by the analysis. BSmooth is one analytical package that &#34;smoothes&#34; the methylation levels across a window of multiple CpGs. For the current experiment, BSmooth implicated a DMR whose methylation levels were validated by bisulfite pyrosequencing. However, there will likely be discrepancies between methylation levels predicted by BSmooth and those verified by pyrosequencing. The discrepancies arise from the smoothing function that estimates the average methylation values across all of the CpGs within a DMR, including consecutive CpGs that may differ in DNA methylation by more than 50% or CpGs whose methylation values were excluded due to sub-threshold read depth. R-packages such as MethylKit24 can be used to identify smaller windows of CpGs or even single CpGs whose methylation levels correlate strongly with those validated by pyrosequencing. Implementing different packages and testing their predicted regions or CpGs of differential methylation by pyrosequencing will ensure robustness of data. Alternatively, original Methyl-Seq libraries can be resequenced and added to the read files to increase read depth. Since determination of methylation levels are semi-quantitative and dictated by the number of reads [(# of CpGs)/(# of TpGs+CpGs)], increasing the read depth for a given CpG will increase the accuracy of its percent methylation value. In this study, we only considered CpGs whose methylation values were determined by at least ten reads and achieved an overall read coverage of 19x for each CpG.
The rat Methyl-Seq platform is not without its limitations. While it is more cost effective than whole-genome bisulfite sequencing, it is considerably more expensive than other methods. Nevertheless, most of the cost was for purchasing lanes on the sequencer and not for the capture system. Depending on the read depth necessary, with cross-tissue comparisons requiring less due to large (25&#8211;70%) differences12 in DNA methylation, the cost can be reduced by multiplexing more samples per lane and using a higher-capacity platform. Also, the sample preparation is more time-consuming than other methods. While similar to other pulldown approaches that incorporate next-generation sequencing, the added bisulfite conversion and purification steps add to the work load. Overall, the Methyl-Seq platform is a cost-effective alternative to whole-genome sequencing and provides base-pair resolution at more than 2.3 million CpGs, which is considerably more than those assayed by microarray-based platforms. To date, the commercially-available human and mouse Methyl-Seq platforms have been used to document alcohol-dependent changes in the macaque brain25,26, neurodevelopmental genes in the mouse brain9, and blood-brain targets of glucocorticoids10. Further, the ability to target specific regions regardless of sequence recognition by restriction enzymes makes it an ideal platform for cross-species comparisons. For this study, we designed the Methyl-Seq platform for the rat, for which many pharmacological, metabolic, and behavioral experiments are performed without the benefit of a genome-wide methylomic tool. Our data show that it can be used to detect DMRs in a rat model of stress and correlated other physiological parameters such as overall plasma CORT levels.
The Methyl-Seq platform is ideal for epigenetic experiments in animals with sequenced genomes that may not have enough experimental evidence documenting regulatory regions. When such regions are made available, additional regions may be custom-designed and attached to the current version. Further, the platform is ideal for comparative genomics, since the target enrichment is not constrained by restriction enzyme recognition. For instance, the promoter region of any gene of interest can be captured regardless of whether it harbors a specific restriction site. Similarly, any regulatory regions, such as those identified in mouse or humans, which are conserved in the genome of interest can be captured.

Advances in high-throughput sequencing have led to a wealth of genomic sequences for both model and non-model organisms. The availability of such sequences has facilitated research in genetics, comparative genomics, and transcriptomics. For instance, available genomic sequences are highly useful for aligning sequencing data from ChIP-Seq experiments that enrich DNA based on its association with histone modifications1, or bisulfite sequencing, which measures DNA methylation by detecting uracil formed from bisulfite conversion of unmethylated cytosines2. However, there have been delays in the implementation of epigenomic platforms that incorporate available genomic sequencing data in their design due to a lack of annotated data of species-specific regulatory sequences that can influence gene function. 
In particular, DNA methylation is one of the most widely studied epigenetic modifications on DNA that can leverage available genomic data for building a methylomic platform. One such example is an array-based platform for the human methylome3, which has been widely used in various disciplines from oncology to psychiatry4,5. Unfortunately, similar platforms for non-human animal models are scarce, as there are virtually no widely-used platforms that have taken advantage of the genomic sequence in their initial design.
A common method to assess the methylomic landscape of non-human animal models is reduced representation bisulfite sequencing (RRBS)6. This approach overcomes the cost of whole-genome bisulfite sequencing that, while providing a comprehensive methylomic landscape, provides lower read-depth coverage due to cost and limited functional information in large gene-poor areas of the genome2. RRBS involves restriction digest and size-selection of genomic DNA to enrich for highly GC-rich sequences such as CpG islands that are commonly found near gene promoters and thought to play a role in gene regulation7. While the RRBS method has been used in a number of important studies, its reliance on restriction enzymes is not without notable challenges and limitations. For instance, enrichment of GC-rich sequences in RRBS is entirely dependent on the presence of specific sequences recognized by the restriction enzyme and subsequent size selection by electrophoresis. This means that any genomic areas that do not contain these restriction sites are excluded during size selection. Also, cross-species comparisons are challenging unless the same restriction sites are present in the same loci among the different species. 
One approach to overcoming the limitations of RRBS is to use an enrichment method that takes advantage of the published genomic sequence in the design of the platform. The array-based human platform uses primer probes designed against specific CpGs for allele-specific (CG vs. TG after bisulfite conversion) target annealing and primer extension. Its design reflects not only the available human genomic sequence, but experimentally-verified regulatory regions acquired from multiple lines of inquiry, such as ENCODE and ENSEMBL8. Despite its wide use in human methylomic investigations, a similar platform does not exist for model animals. In addition, the array-based format places significant constraint on the surface area available for probe placement. In the past several years, efforts have been made to combine the target-specificity afforded by capture probe design and the high-throughput feature of next-generation sequencing. Such an endeavor has resulted in the sequencing-based target enrichment system for the mouse genome (mouse Methyl-Seq), which was used to identify brain-specific or glucocorticoid-induced differences in methylation9,10. Similar platforms for other model and non-model animals are needed to facilitate epigenomic research in these animals.
Here, we demonstrate the implementation of this novel platform to conduct methylomic analysis on the rat. The rat has served as an important animal model in pharmacology, metabolism, neuroendocrinology, and behavior. For example, there is an increasing need to understand the underlying mechanisms that give rise to drug toxicity, obesity, stress response, or drug addiction. A high-throughput platform capable of capturing methylomic changes associated with these conditions would increase our understanding of the mechanisms. Since the rat genome still lacks annotation for regulatory regions, we incorporated non-redundant promoters, CpG islands, island shores11, and previously identified GC-rich sequences into the rat Methyl-Seq platform12. 
To assess successful design and implementation of the SureSelect Target Enrichment (generically referred to as Methyl-Seq) platform for the rat genome, we employed a rat model of chronic variable stress (CVS)13 to identify differentially methylated regions between unstressed and stressed animals. Our platform design, protocol, and implementation may be useful for investigators who may want to conduct a comprehensive and unbiased epigenetic investigation on an organism whose genomic sequence is already available but remains poorly annotated.

<table><tbody><tr><td>Radioimmuno assay (RIA)</td><td>MP Biomedicals</td><td>7120126</td><td>Corticosterone, 125I labeled</td></tr><tr><td>Master Pure DNA Purification Kit</td><td>Epicentre/Illumina</td><td>MC85200</td><td></td></tr><tr><td>Thermal-LOK 2-Position Dry Heat Bath</td><td>USA Scientific</td><td>2510-1102</td><td>Used with 1.5 mL tubes</td></tr><tr><td>Vortex Genie 2</td><td>Fisher</td><td>12-812</td><td>Vortex Mixer</td></tr><tr><td>Ethyl alcohol, Pure</td><td>Sigma-Aldrich</td><td>E7023</td><td>100% Ethanol, molecular grade</td></tr><tr><td>Centrifuge 5424 R</td><td>Eppendorf</td><td>-</td><td>Must be capable of 20,000 x &lt;em&gt;g&lt;/em&gt;</td></tr><tr><td>Qubit 2.0</td><td>ThermoFisher Scientific</td><td>Q32866</td><td>Fluorometer</td></tr><tr><td>Qubit dsDNA BR Assay Kit</td><td>ThermoFisher Scientific</td><td>Q32850</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>ThermoFisher Scientific</td><td>Q32851</td><td>High sensitivity DNA detection reagents</td></tr><tr><td>Qubit Assay Tubes</td><td>ThermoFisher Scientific</td><td>Q32856</td><td></td></tr><tr><td>SureSelectXT Rat Methyl-Seq Reagent Kit</td><td>Agilent Technologies</td><td>G9651A</td><td>Reagents for preparing the Methyl-Seq library</td></tr><tr><td>SureSelect Rat Methyl-Seq Capture Library</td><td>Agilent Technologies</td><td>931143</td><td>RNA baits for enrichment of rat targets</td></tr><tr><td>IDTE, pH 8.0</td><td>IDT DNA</td><td>11-05-01-09</td><td>10 mM TE, 0.1 mM EDTA</td></tr><tr><td>DNA LoBind Tube 1.5 mL</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>Covaris E-series or S-series</td><td>Covaris</td><td>-</td><td>Isothermal sonicator</td></tr><tr><td>microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16mm (25)</td><td>Covaris</td><td>520045</td><td></td></tr><tr><td>Water, Ultra Pure (Molecular Biology Grade)</td><td>Quality Biological</td><td>351-029-721</td><td></td></tr><tr><td>Veriti 96 Well-Thermal Cycler</td><td>Applied Biosystems</td><td>4375786</td><td></td></tr><tr><td>AMPure XP Beads</td><td>Beckman Coulter</td><td>A63880</td><td>DNA-Binding magnetic beads</td></tr><tr><td>96S Super Magnet</td><td>ALPAQUA</td><td>A001322</td><td>Magnetic plate for purification steps</td></tr><tr><td>2200 TapeStation</td><td>Agilent Technologies</td><td>G2965AA</td><td>Electrophresis-based bioanalyzer</td></tr><tr><td>D1000 ScreenTape</td><td>Agilent Technologies</td><td>5067-5582</td><td></td></tr><tr><td>D1000 ScreenTape High Sensitivity</td><td>Agilent Technologies</td><td>5067-5584</td><td></td></tr><tr><td>D1000 Reagents</td><td>Agilent Technologies</td><td>5067-5583</td><td></td></tr><tr><td>D1000 Reagents High Sensitivity</td><td>Agilent Technologies</td><td>5067-5585</td><td></td></tr><tr><td>DNA110 SpeedVac</td><td>ThermoFisher Scientific</td><td>-</td><td>Vacuum Concentrator</td></tr><tr><td>Dynabeads MyOne Streptavidin T1 magnetic beads</td><td>Invitrogen</td><td>65601</td><td>Streptavidin magnetic beads</td></tr><tr><td>Labquake Tube Rotator</td><td>ThermoFisher Scientific</td><td>415110Q</td><td>Nutator Mixer is also acceptable</td></tr><tr><td>EZ DNA Methylation-Gold Kit</td><td>Zymo Research</td><td>D5006</td><td>Bisulfite conversion kit. Contains Binding, Wash, Desulphonation, and Elution buffers</td></tr><tr><td>Illumina Hi-Seq 2500</td><td>Illumina</td><td>-</td><td>Next-generation sequencing machine</td></tr><tr><td>PCR and Pyrosequencing Primers</td><td>IDT DNA</td><td>Variable</td><td></td></tr><tr><td>Taq DNA Polymerase with ThermoPol Buffer - 2,000 units</td><td>New England BioLabs</td><td>M0267L</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) Solution Set</td><td>New England BioLabs</td><td>N0446S</td><td></td></tr><tr><td>Pyromark MD96</td><td>QIAGEN</td><td>-</td><td>Pyrosequencing machine</td></tr><tr><td>Ethyl Alcohol 200 Proof</td><td>Pharmco-Aaper</td><td>111000200</td><td>70% Ethanol solution</td></tr><tr><td>Sodium Hydroxide Pellets</td><td>Sigma-Aldrich</td><td>221465</td><td>0.2 M NaOH denature buffer solution</td></tr><tr><td>Tris (Base) from J.T. Baker</td><td>Fisher Scientific</td><td>02-004-508</td><td>10 mM Tris Acetate Buffer wash buffer solution</td></tr><tr><td>PyroMark Gold Q96 Reagents (50x96)</td><td>QIAGEN</td><td>972807</td><td>Reagents required for pyrosequencing</td></tr><tr><td>PyroMark Annealing Buffer</td><td>QIAGEN</td><td>979009</td><td></td></tr><tr><td>PyroMark Binding Buffer (200 mL)</td><td>QIAGEN</td><td>979006</td><td></td></tr><tr><td>Streptavidin Sepharose High Performance Beads</td><td>GE Healthcare</td><td>17-5113-01</td><td>Streptavidin-coated sepharose beads</td></tr><tr><td>PyroMark Q96 HS Plate</td><td>QIAGEN</td><td>979101</td><td>Pyrosequencing assay plate</td></tr><tr><td>Eppendorf Thermomixer R</td><td>Fisher Scientific</td><td>05-400-205</td><td>Plate mixer. 96-well block sold separately (cat. No 05-400-207)</td></tr><tr><td>SureDesign Website</td><td>Agilent Technologies</td><td>-</td><td>Target capture design software (https://earray.chem.agilent.com/suredesign/)</td></tr><tr><td>UCSC Genome Browser</td><td>University of California Santa Cruz</td><td>-</td><td>rat Nov 2004 rn4 assembly</td></tr><tr><td>Agilent Methyl-Seq Protocol</td><td>Agilent Technologies</td><td>-</td><td>https://www.agilent.com/cs/library/usermanuals/public/G7530-90002.pdf</td></tr></tbody></table>

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.

Watch this Scientific Journal Video about A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure at JoVE.com

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

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 ...

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10.7K Views.  Johns Hopkins School of Medicine. 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.

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

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

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

Neuroscience

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 &#177; 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 &#177; 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid ...

Genetics

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 ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Biology

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.

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.

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...

Modeling Early-Life Adversity in Mice Using Limited Bedding and Nesting

Limited Bedding and Nesting as a Model for Early-Life Adversity in Mice

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing neuropsychiatric disorders such as depression. Animal models for ELA have been used to study the impact of chronic stress on brain development, and typically rely on manipulating the quality and/or quantity of maternal care, as this is the major source of early-life experiences in mammals, including humans. Here, a detailed protocol for employing the Limited Bedding and Nesting (LBN) model in mice is provided. This model mimics a low-resource environment, which provokes fragmented and unpredictable patterns of maternal care during a critical developmental window (postnatal days 2-9) by limiting the amount of nesting materials given to the dam to build a nest for her pups and separating the mice from the bedding via a mesh platform in the cage. Representative data are provided to illustrate the changes in maternal behavior, as well as the diminished pup weights and long-term changes in basal corticosterone levels, that result from the LBN model. As adults, offspring reared&#160;in the LBN environment have been shown to exhibit an aberrant stress response, cognitive deficits, and anhedonia-like behavior. Therefore, this model is an important tool to define how the maturation of stress-sensitive brain circuits is altered by ELA and results in long-term behavioral changes that confer vulnerability to mental disorders.

Author Spotlight: Exploring Microglial Interactions with Stress-Response Circuitry Using the Limited Bedding and Nesting Model

This protocol describes an animal model for studying how early-life adversity, provoked by an impoverished environment and unpredictable maternal care during the early postnatal period, affects brain development and the future risk of mental disorders.

Early-life adversity (ELA), such as abuse, neglect, lack of resources, and an unpredictable home environment, is a known risk factor for developing ...

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 ...