This work was funded by the Max Planck Institute of Psychiatry and the European Union's Marie Curie Initial Training Network (NINA Contract 238665).

1. Programming by Early Life Adversity
Maternal separation (MS) is performed to induce early-life stress (ELS) in pups delivered by timed-pregnant C57BL/6N mice (postnatal day 0 (P0) on day of birth).
<ol>
 <li>Individual litters are placed in clean cages (with heating pad) for 3 h daily from P1-10.</li>
 <li>Control (non-ELS) pups remain undisturbed in the maternal nest throughout.</li>
 <li>Pups are kept with their mothers until weaning (P21), after which time they are housed in sex-matched groups (3-5 mice per cage) under standard laboratory animal housing conditions.</li>
</ol>
2. Isolation and Dissection of Brain Tissue
<ol>
 <li>Mice are killed at desired ages by cervical dislocation. Note: because this protocol involves a stress paradigm, no anesthesia is given prior to cervical dislocation, to avoid interfering with normal physiological regulation of stress hormones. Skulls are opened and brains are carefully removed and immediately snap-frozen by immersion in iso-pentane-dry ice, before being stored at -80 &deg;C.</li>
 <li>Brains are cryosectioned (10 &mu;m thickness); sections are mounted on Superfrost glass slides and maintained at -20 &deg;C. Reference to a standard mouse stereotaxic atlas (e.g. Paxinos 6) is used to verify anatomical precision and to ensure inclusion of the region of interest (e.g. for neurons in the paraventricular nucleus - PVN - collect sections starting at the level of the rostral PVN, bregma -0.75 to -0.85).</li>
 <li>Sections are stained with cresyl violet to facilitate identification of different brain structures. Punches (0.8 mm) of the regions of interest (e.g. PVN) are obtained by in loco microdissection.</li>
</ol>
3. Nucleic Acid Extraction from Brain Punches
An optimized protocol for simultaneously extracting DNA and RNA from tiny neuroanatomically-defined brain regions for bisulfite and gene expression analyses is described 7.
Note: Considering that RNA is less stable than DNA in the GTC-Buffer, we recommend processing RNA first. The homogenate used for DNA purification can be kept at room temperature (RT) during that time.
<ol>
 <li>Homogenize punches using a pipette and vortexer in 400 &mu;l of guanidinium thiocyanate (GTC) buffer (4.5 M guanidinium thiocyanate, 2% N-lauroylsarcosine, 50 mM EDTA pH 8.25 mM Tris-HCl pH 7.5, 0.1 M beta-mercaptoethanol, 0.2% antifoam A) at RT, passing several times through a hypodermic syringe (29G) (Figure 2).</li>
 <li>Split lysate into equal parts; both RNA and DNA may be extracted at the same time or separately, depending on particular experimental needs.</li>
 <li>For RNA purification add 1/10 volume of NaOAc, 1 volume AquaPhenol (Appligene) (pH 4) and 1/2 volume of chloroform:isoamyl (24:1) to lysate. Vortex vigorously after each step and incubate on ice for 10 min, centrifuge (20 min at 10,000 g at 4 &deg;C); add an equal volume of 70% EtOH to aequous phase.</li>
 <li>Transfer mixture to an RNA spin column (e.g. Nucleospin RNA II from Macherey-Nagel) and perform on-column DNase-digestion and washing steps (follow manufacturer's protocol); elute RNA in 25 &mu;l H2O.</li>
 <li>For DNA purification an optimized protocol of the Qiagen DNeasy Blood and Tissue Kit is used. Equilibrate lysate with equal volumes of Buffer AL and 100% EtOH, load on a Spin Column centrifuge (1 min at 10,000 g at RT) and discard flow-through.</li>
 <li>Add 500 &mu;l Buffer AW1 including 5 &mu;l RNAse (1 mg/ml) to column and incubate 10 min at RT. Spin (1 min at 10,000 g, RT) and discard flow-through.</li>
 <li>Wash column with 500 &mu;l Buffer AW2, discard flow-through and spin-dry empty column (1 min at 15,000 g).</li>
 <li>Add prewarmed (70 &deg;C) Buffer AE and incubate columns for 10 min at 70 &deg;C. Elute by centrifugation (1 min, 10,000 g), re-apply flow-through and repeat centrifugation step.</li>
</ol>
Use a spectrophotometer to determine DNA and RNA concentrations. A typical PVN punch yields ~600 ng DNA and ~400 ng RNA. For gene expression analysis by quantitative PCR (qPCR), we typically use ~100 ng of RNA in the reverse transcription reaction.
4. Bisulfite Conversion
Sodium Bisulfite is used to convert non-methylated cytosines to uracils. In contrast, methylated cytosines are protected from conversion. Therefore, all cytosines that are detected in the final sequencing of the bisulfite PCR-amplicon represent methylated cytosines.
Bisulfite conversion causes substantial DNA degradation which can be a limiting factor in PCR analysis. Optimized reaction conditions that maximize cytosine conversion, reduce DNA fragmentation and maintain single-stranded DNA even at lower temperatures can be achieved using Qiagen's EpiTect Bisulfite Kit. In our hands ~200 ng of DNA purified from micropunches provide a sufficient amount of starting material for the bisulfite reaction.
To prevent differences in the efficiency of the conversion reaction, the amount of template DNA should be kept constant among all samples processed during an experiment. In addition, sequence reads should be scrutinized for conversion efficiency by examining the rate of non-converted non-CpGs. This rate should exceed 98%. Lower values indicate incomplete or inefficient bisulfite conversion and the underlying sequences should be excluded from the analysis.
5. Bisulfite PCR
<ol>
 <li>Design bisulfite sequencing primers that are specific to bisulfite converted DNA (see discussion); Methyl Primer Express software (<a href="https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&amp;catID=602121" target="_blank">https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&amp;catID=602121</a>) will aid this and help to determine optimal annealing temperatures in pilot experiments.</li>
 <li>Prepare PCR-Master-Mix (for one reaction) as follows:
 2.5 &mu;l 10x PCR buffer 
 0.5 &mu;l 10 mM dNTPs 
 1 &mu;l 10 &mu;M forward primer 
 1 &mu;l 10 &mu;M reverse primer 
 0.125 &mu;l Qiagen Hotstart Taq Plus 
 fill up to 23 &mu;l with H2O </li>
 <li>Add 2 &mu;l bisulfite-treated DNA to reaction.</li>
 <li>Amplify using the following conditions:
 1 cycle 6 min 95 &deg;C 
 45-50 cycles of 1 min 95 &deg;C, 1 min at optimal annealing temperature, 1 min 72 &deg;C 
 1 cycle 5 min 72 &deg;C </li>
</ol>
Analyze 7 &mu;l of PCR product by agarose gel electrophoresis to verify size of the amplicon and purify remaining PCR reaction for subsequent ligation using a commercially available PCR clean-up kit (e.g. Macherey-Nagel Nucleospin Extract). In case additional undesired PCR products are obtained, gel purification is recommended.
6. Bisulfite Sequencing
High-resolution DNA methylation profiles deduced from single clone readings can detect small changes in DNA methylation and identify regulatory regions that are responsive to treatment (environmental programming). The bisulfite sequencing process comprises three consecutive working steps. Firstly, PCR products obtained by prior bisulfite PCR are ligated into a vector and transformed into bacteria. Secondly, colony PCR from single clones is employed to determine the correct size of the insert. Thirdly, positive colony PCRs are cleaned up and subjected to Big-Dye sequencing reaction. Following a clean-up step, products are electrophoresed on a capillary sequencer.
6.1 Ligation and Transformation
Note: We routinely use the pGEM-T vector cloning kit (Promega). In our experience, the cloning efficiency depends critically on the insert to be ligated. Different vectors should be tested in case low numbers of recombinant clones are repeatedly obtained.
<ol>
 <li>Set up ligation reaction:
 5 &mu;l 2x Ligation Buffer 
 1 &mu;l pGEM-T Vector 
 1 &mu;l T4-Ligase 
 3 &mu;l cleaned-up PCR product </li>
 <li>Mix reaction by pipetting and incubate over night at 4 &deg;C.
 Note: Ligation may also be carried out for 1 h at RT as well. To increase number of recombinant clones, over night ligation at 4 &deg;C is preferred. </li>
 <li>Clean-up of ligation products by ethanol precipitation:
 <ol class="lalpha">
 <li>Add 1 &mu;l glycogen, 1 &mu;l 3M NaAc and 25 &mu;l 100% EtOH to the ligation reaction and precipitate for 1 min in liquid nitrogen.</li>
 <li>Centrifuge (15,0000 g for 30 min at 4 &deg;C) and discard supernatant.</li>
 <li>Wash with 300 &mu;l 70% EtOH and centrifuge (15,000 g for 20 min at 4 &deg;C), discard supernatant and dry pellet at RT (10 min).</li>
 <li>Resuspend pellet in 10 &mu;l H2O.</li>
 </ol>
 </li>
</ol>
6.2 Transformation of ligation products in electrocompetent bacteria
<ol>
 <li>Precool electroporation cuvettes (1 mm width) on ice and thaw aliquot of electrocompetent DH5&alpha; bacteria on ice.</li>
 <li>Add 45 &mu;l DH5&alpha; bacteria to 10 &mu;l cleaned up ligation product (see above) and transfer to cuvette.</li>
 <li>Transform bacteria at 1.5 kV, 200 &Omega;, 15 &mu;F and add 1 ml of prewarmed SOB medium directly after pulse delivery.</li>
 <li>Recover bacteria for 1 h at 37 &deg;C, spread 100 &mu;l of suspension on LB/ampicillin plates coated with IPTG/X-Gal.</li>
 <li>Incubate plates overnight at 37 &deg;C.</li>
</ol>
6.3 Colony PCR
Note: Colony PCR from single clones is conducted to ensure that inserts are of predicted size. Delayed color development from the blue/white screening, undesired recombination events during ligation, incorporation of oligomeric primer pairs or truncated PCR products may otherwise lead to faulty sequencing results. We routinely use T7 and SP6 primers to amplify cloned inserts; this approach results in additional vector sequences of approximately 150 bp. T7 primer is used in the sequencing reaction in a later step.
<ol>
 <li>Set up colony PCR reaction in 96-well plate. For one reaction use:
 3 &mu;l 2.5 mM MgCl2 
 2.5 &mu;l 10x Taq buffer 
 1.5 &mu;l 10 mM dNTP 
 2 &mu;l 2.5 mM T7 primer 
 2 &mu;l 2.5 mM SP6 primer 
 1 &mu;l Fermentas Taq Polymerase 
 fill up to 25 &mu;l with H2O </li>
 <li>Dispense 25 &mu;l/well of master mix into each well of a 96-well plate.</li>
 <li>Pick positive (white) clone from plate with pipette tip and dip into PCR reaction.</li>
 <li>Amplify using following conditions:
 1 cycle 4 min at 95 &deg;C 
 10 cycles 30 s at 94 &deg;C, 30 s at 56 &deg;C and 30 s at 72 &deg;C 
 30 cycles 30 s at 94 &deg;C, 30 s at 48 &deg;C and 30 s at 72 &deg;C 
 1 cycle 5 min at 72 &deg;C </li>
 <li>Load 5 &mu;l of colony PCR on an agarose gel and determine reactions that contain the right insert size.</li>
 <li>Colony PCRs containing the desired amplicons are cleaned up using a commercially available kit (Machery Nagel Nucleofast).</li>
</ol>
6.4 Big-Dye terminator reaction and sequencing
<ol>
 <li>Prepare Master mix for Big-Dye reaction. For one reaction use:
 1 &mu;l H2O 
 0.5 &mu;l Big-Dye reagent 
 1.5 &mu;l Sequencing buffer </li>
 <li>Dispense 3 &mu;l/well into each well of a 96-well plate, to each well add 2 &mu;l of cleaned up colony PCR product and run reaction on a thermocycler with the following parameters:
 1 cycle 1 min at 96 &deg;C 
 35 cycles of 10 s at 96 &deg;C, 5 s at 50 &deg;C and 4 min at 60 &deg;C </li>
 <li>The Big-Dye reaction is cleaned up using a commercial kit (Millipore Montage 96 Sequencing Clean-Up Kit) and processed on a capillary sequencer (e.g. ABI 3100 DNA).</li>
 <li>Sequences are analyzed using the Biq Analyzer (<a href="http://biq-analyzer.bioinf.mpi-sb.mpg.de/" target="_blank">http://biq-analyzer.bioinf.mpi-sb.mpg.de/</a>) or the online tool BISMA (<a href="http://biochem.jacobs-university.de/BDPC/BISMA/" target="_blank">http://biochem.jacobs-university.de/BDPC/BISMA/</a>) to derive the methylation pattern of the investigated DNA region (Figure 4).</li>
</ol>
7. Representative Results
To gain insight into the influence of ELS on the Avp expression and methylation status, C57BL/6N mice were processed according to the workflow described above. Briefly, a group of C57BL/6N mice was subjected to ELS while the control group was left undisturbed. The PVN and the supraoptic nucleus (SON) were punched and DNA and RNA were isolated simultaneously from the single punches. RNA was reverse transcribed and the levels of Avp transcripts were measured by qPCR analysis and normalized to the expression of Hprt and Gapdh housekeeping genes. DNA was bisulfite treated, amplified with primers specific to the Avp enhancer (Figure 4a) and the PCR products were cloned and sequenced. At least 20 recombinant clones from each mouse/PCR were analyzed to determine methylation frequencies for the CpGs contained in the PCR amplicon (Figure 4b). Compared to controls, ELS induced a significant hypomethylation at CpG10, CpG12, CpG13 and Cpg14 of the Avp enhancer (p &lt; 0.05, n = 6-8 animals) suggesting epigenetic marking of this regulatory region through early-life experiences. In contrast to the PVN, methylation of the Avp enhancer was unaffected by ELS in the SON illustrating tissue specificity of epigenetic marking (Figure 4c). Analysis of the DNA methylation status at CpG10 and Avp gene expression in control animals (n = 6) evidenced a negative correlation pointing to a role of DNA methylation in the fine-tuning of AVP gene expression (Figure 4d).
<img alt="Figure 1" src="/files/ftp_upload/3938/3938fig1.jpg" /> 
Figure 1. Micropunches are obtained from dissected brains from control and early-life stressed mice. Following DNA and RNA isolation, gene expression is determined by qRT-PCR while bisulfite treated DNA is amplified and purified products are cloned in a suitable vector allowing identification of recombinant clones by blue/white selection. Correct insert sizes are verified by colony PCR prior to carrying out Big-Dye reaction and processing on a capillary sequencer. Methylation pattern are visualized by appropriate software tools. <a href="https://www.jove.com/files/ftp_upload/3938/3938fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3938/3938fig2.jpg" /> 
Figure 2. Timeline from DNA/RNA extraction to bisulfite sequencing.
<img alt="Figure 3" src="/files/ftp_upload/3938/3938fig3.jpg" /> 
Figure 3. Workflow for the simultaneous extraction of DNA and RNA. The punch of the hypothalamic nucleus paraventricularis, a tiny Avp gene expressing brain region, is placed in 400 &mu;l GTA buffer, vortexed until disrupted and further homogenized by passing through a syringe (29G). The homogenate is then split for the purification of RNA and DNA. The split can be 1:1 or of different proportions depending on the particular experimental question. Homogenates should be processed within a few hours.
<img alt="Figure 4" src="/files/ftp_upload/3938/3938fig4.jpg" /> 
Figure 4. CpG methylation at the Avp locus in 6 weeks old control and ELS animals. (A) Scheme of the Avp and oxytocin genes orientated tail to-tail and separated by the intergenic region (IGR). Exons are depicted by open (numbered) boxes. The distribution of CpG residues is indicated and size and position of the respective PCR amplicon containing CpG 10 to CpG14 is marked by a bold line. (B) Methylation of the Avp enhancer in the PVN in control and ELS animals (n = 6-8). (C) Methylation of the Avp enhancer in the SON in control and ELS animals (n = 8-10). (D) Avp gene expression was correlated with methylation at CpG10 (n = 6). <a href="https://www.jove.com/files/ftp_upload/3938/3938fig4large.jpg" target="_blank"> Click here to view larger figure</a>.

<ol>
	<li>Jirtle, R. L., Skinner, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+epigenomics+and+disease+susceptibility.">Environmental epigenomics and disease susceptibility.</a> Nat. Rev. Genet. 8, 253-262 (2007).</li><li>Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+programming+of+the+HPA+axis:+Early+life+decides.">Epigenetic programming of the HPA axis: Early life decides.</a> Stress (Amsterdam, Netherlands). , (2011).</li><li>Gluckman, P. D., Hanson, M. A., Buklijas, T., Low, F. M., Beedle, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+mechanisms+that+underpin+metabolic+and+cardiovascular+diseases.">Epigenetic mechanisms that underpin metabolic and cardiovascular diseases.</a> Nat. Rev. Endocrinol. 5, 401-408 (2009).</li><li>Murgatroyd, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+DNA+methylation+programs+persistent+adverse+effects+of+early-life+stress.">Dynamic DNA methylation programs persistent adverse effects of early-life stress.</a> Nat. Neurosci. 12, 1559-1566 (2009).</li><li>Murgatroyd, C., Wu, Y., Bockm&#252;hl, Y., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+learn+from+stress:+How+infantile+trauma+programs+us+for+depression.">Genes learn from stress: How infantile trauma programs us for depression.</a> Epigenetics. 5, (2010).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates, Compact: The Coronal Plates and Diagrams: Compact. The Coronal Plates and Diagrams. , (2008).</li><li>Bettscheider, M., Murgatroyd, C., Spengler, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+DNA+and+RNA+isolation+from+brain+punches+for+epigenetics.">Simultaneous DNA and RNA isolation from brain punches for epigenetics.</a> BMC research notes. 4, 314 (2011).</li><li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Reed, K., Poulin, M. L., Yan, L., Parissenti, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+bisulfite+sequencing+PCR+with+pyrosequencing+for+measuring+differences+in+DNA+methylation.">Comparison of bisulfite sequencing PCR with pyrosequencing for measuring differences in DNA methylation.</a> Anal. Biochem. 397, 96-106 (2010).</li></ol>

No conflicts of interest declared.

Epigenetic programming is increasingly recognized as an important mechanism underlying health and disease. Here we present a refined method for the simultaneous isolation of DNA and RNA from micropunches and the subsequent steps to obtain DNA methylation profiles through bisulfite sequencing. The optimized workflow allows convenient analysis of epigenetic programming in the brain in response to environmental stimuli. Moreover, this approach facilitates the investigation of the functional relationship between DNA methylation and gene expression as both are analyzed from the same sample. Finally, animal numbers needed for the experiment can be significantly reduced.
Certain precautions and guidelines should be followed while performing the presented workflow. Considering the extraordinary complexity and heterogeneity of the brain and since methylation and expression pattern may vary in a cell- and tissue specific manner it is of utmost importance that micropunching is performed in a standardized manner. As a case in point, epigenetic programming of Avp is detected in the paravaventriuclar, but not in the supraoptic nucleus (Figure 4b-d), two Avp expressing regions of the hypothalamus 4. In this respect, availability of DNA and RNA from the same tissue punch is advantageous as RNA expression of certain tissue specific marker genes can be used in some cases to certify that the correct area was punched. Although micropunching can reduce cellular heterogeneity, it has to be remembered that this approach does not lead to the isolation of single cell types or populations. Additional purification steps might be considered to address this topic.
For bisulfite sequencing optimal primer design for PCR amplification following bisulfite treatment is essential. PCR products exceeding 400 bp in length can be problematic due to the degradation of DNA by the bisulfite treatment. It should be also noted that nested PCR can give biased results if only a few intact templates contribute to the amplification process. Primer pairs that are specific for the modified amplicon should be designed; their sequences should not contain CpG dinucleotides in order to avoid methylation-biased amplification. In addition, primers should contain non-CpG cytosines to prevent amplification of unmodified or incompletely converted DNA. Amplification of some templates may benefit from conducting Hot-start PCR. In any case, suitability of primer pairs should be verified in pilot experiments so as to prevent spoilage of valuable experimental specimens.
Bisulfite sequencing represents a standard method for site-specific methylation analysis as it is able to reliably and precisely detect the methylation of single CpG residues in an allele-specific manner 8. Direct sequencing of the PCR product is not advised as mixed methylation of a CpG residue will appear as a C/T double peak in the electropherogram, which can prevent quantification of methylation at the site concerned. For this reason an intermediate cloning step is conducted and several clones are sequenced (~20-30) to determine the extent of methylation. Pyrosequencing represents an alternative method to quantify DNA methylation. It also utilizes bisulfite conversion and bisulfite PCR but instead of cloning and sequencing the amplicon is subjected to a direct pyrosequencing reaction where the methylation status of a CpG residue is read as a C/T single nucleotide polymorphism that can be easily quantified. Both methods detect similar levels of methylation 9 but since the cloning-step can be omitted pyrosequencing is more time-efficient. However, depending on the template only 40-100 nucleotides can be sequenced at once so that several rounds of pyrosequencing with different sequencing primer are necessary. This is a critical limitation, given that higher amounts of DNA (about 1 &mu;g per reaction) are required, which foreseeable exceed the amounts available from tiny brain tissue punches. Thus initial scanning of regions in the range of several kilobases by pyrosequencing appears less suited as single clone reading. Nevertheless, following on identification of a small region of interest pyrosequencing should be considered.
Collectively, the protocol described above provides an accurate, efficient and streamlined approach for the rapid and cost-effective analysis of brain micropunches for epigenetic changes. Multiple specimens can be processed in a single run and the methods are suitable when running samples from intermediate-sized experiments.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>DNeasy Blood &amp; Tissue Kit</td>
 <td>Qiagen</td>
 <td>69506</td>
 </tr>
 <tr>
 <td>Nucleospin RNA II</td>
 <td>Macherey-Nagel</td>
 <td>740955.50</td>
 </tr>
 <tr>
 <td>Epitect Bisulfite Kit</td>
 <td>Qiagen</td>
 <td>59104</td>
 </tr>
 <tr>
 <td>Nucleospin Extract</td>
 <td>Macherey-Nagel</td>
 <td>740609.50</td>
 </tr>
 <tr>
 <td>pGEM-T Vector Cloning Kit</td>
 <td>Promega</td>
 <td>A3600</td>
 </tr>
 <tr>
 <td>Nucleofast 96</td>
 <td>Macherey-Nagel</td>
 <td>743100.50</td>
 </tr>
 <tr>
 <td>Montage 96 Sequencing Clean-Up</td>
 <td>Millipore</td>
 <td>LSK09624</td>
 </tr>
 <tr>
 <td>Hotstar Taq Polymerase</td>
 <td>Qiagen</td>
 <td>203203</td>
 </tr>
 <tr>
 <td>Taq Polymerase</td>
 <td>Fermentas</td>
 <td>EP0401</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain at JoVE.com

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

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 ...

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18.7K Views.  Max Planck Institute of Psychiatry. 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.

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

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...