This work was supported by R01NS075062-01A1. FACS sorting performed at UAB Comprehensive Flow Cytometry Core facility (P30 AR048311, P30 A1027767). Dr. Scott Philips from the UAB Neurobiology Core facility and Dr. Susan Nozell from UAB CDIB assisted with technical aspects of the luciferase assay.

All animals were handled in accordance with the National Institutes of Health guidelines. The Animal Care and Use Committee at the University of Alabama at Birmingham approved animal use.
1. Obtaining RNA and DNA from an Enriched Astrocytic Population using Fluorescent Activated Cell Sorting (FACS) of Astrocytes from Whole Brain Tissue
<ol>
	<li>Sedate animal with CO2 for 1 min and then rapidly decapitate. Dissect cortices as described in Albuquerque et al.,26; it is not necessary to remove meninges. 
	Note: Transgenic rats expressing eGFP under the S100&#946; (an astrocyte marker) promoter were generated by Itakura et al.27 and utilized for FACS sorting of astrocytes.</li>
	<li>Prepare whole brain homogenate using a papain dissociation kit under non-sterile conditions according to manufacturer&#8217;s protocol. Heat-activate papain at 37 &#176;C in water bath for 10 min. Note: Papain solution is provided by manufacturer and contains L-cysteine and EDTA (See Table of Materials).
	<ol>
		<li>Cut or dremel a hole into the top of a 50 ml conical tube to allow tubing carrying 95%O2:5% CO2 to be fed into a closed conical tube (Figure 1A). Place dissected cortices in 10mm culture dish containing dissociation media (MEM supplemented with 20mM glucose and penicillin/streptomycin (500 U/ml) and equilibrated to 95%O2:5% CO2) and use a clean razor blade to mince tissue into 1 x 1 mm2 pieces.</li>
	</ol>
	</li>
	<li>Transfer tissue using 10 ml&#160;manual pipetman to 50 ml conical tube containing papain solution. Allow tissue to settle to bottom of transfer pipette before discharging to minimize the amount of dissociation media carried over. Keep papain solution equilibrated to 95%O2:5% CO2 via surface gas exchange for the duration of the incubation. Do not bubble papain solution. Incubate tissue for 20 min&#160;in 37 &#176;C water bath.</li>
	<li>Following incubation in papain solution, triturate tissue 10 times with a 10 ml&#160;transfer pipette at slow speed. Centrifuge cloudy cell suspension at 1,000 x g for 5 min&#160;at RT.
	<ol>
		<li>Equilibrate DNase/albumin inhibitor solution (provided by manufacturer) via surface gas exchange and re-suspend pelleted cells in 3 ml of DNase/albumin inhibitor solution. Prepare commercial discontinuous density gradient following manufacturer instructions.</li>
	</ol>
	</li>
	<li>Spin discontinuous density gradient at 1,000 x g for 6 min. Isolate dissociated cells from the bottom of tube by sucking bottom pellet using a pipette.</li>
	<li>Re-suspend dissociated cells in 2-3 ml of DPBS with 0.02% bovine serum albumin and 1 mg/ml DNase or preferred HEPES buffered culture media. Pass through 40 &#181;m filter before FACS (Figure 2A). Keep cells on ice until sorting.
	<ol>
		<li>Perform FACS28.</li>
		<li>Pellet cells in 1.5 ml centrifuge tubes at 2,000 x g for 5 min&#160;at 4 &#176;C. 
		Note: Pelleted astrocytes can be utilized immediately or kept in -80 &#176;C until DNA extraction.</li>
	</ol>
	</li>
	<li>Extract RNA and DNA using preferred isolation method 2930. Assess RNA and DNA concentration and quality via spectrophotometer and bioanalyzer 31. 
	Note: Utilize only high quality RNA and DNA in subsequent steps, 260/280=2.0-2.2 and 1.8-1.9, respectively. Bioanalyzer analysis is essential to assess for RNA degradation or DNA fragmentation. RNA or DNA can be utilized immediately or stored in -80 &#176;C or -20 &#176;C, respectively for subsequent studies.</li>
</ol>
2. Assessing DNA Methylation Status of a Gene using Methylation-sensitive High Resolution Melt Analysis (MS-HRMA)
<ol>
	<li>Enter gene sequence of interest into preferred online methylation mapping software to identify any CpG islands in gene of interest 32.
	<ol>
		<li>Design primers against bisulfite converted DNA sequence33 and amplify using preferred DNA polymerase according to manufacturer&#8217;s protocol. Verify amplified product size by running a 1% agarose DNA gel at 100 V for 45 min. Store primers at stock concentration of 20 &#181;M in -20 &#176;C.</li>
	</ol>
	</li>
	<li>Bisulfite convert 500-1,000 ng of DNA of each sample and methylated DNA standards ranging from 0-100% from the same animal species 34. Elute samples to provide a concentration of 20 ng/&#181;l. Verify concentration of bisulfite converted DNA via spectrophotometer 31.</li>
	<li>Setup 20 &#181;l reactions for MS-HRM amplification using preferred DNA polymerase and primers at 5 &#181;M concentration according to Table 1 and 2. Run all samples, including FACS DNA and methylated standards, in triplicate.</li>
	<li>Depending on analysis software, set pre- and post- start and stop parameters around the transitions of the melt curve. Set pre-melt start and pre-melt stop parameters so the difference between the two is 0.2 &#8211; 0.5 &#176;C. Set post-melt start and post-melt stop similarly. Extract peak temperature difference data for each sample.</li>
	<li>Using percent methylated standards (y-value) and their corresponding average peak temperature differences (x-value) generate a linear regression equation (Figure 3A-B, Table 3-4). Use this linear regression equation to estimate methylation status of unknown samples 35.</li>
</ol>
3. Assessing Hyper-methylated Promoter Activity via Use of Luciferase Assay
<ol>
	<li>Identify CpG islands of target gene (Step 2.1). PCR amplify regions of interest 36 and clone upstream of the luc2 Firefly luciferase reporter gene to produce CpG-island-luc2 plasmids 37.</li>
	<li>Linearize 30 &#181;g of CpG-island-luc2 plasmid via restriction enzyme digestion 38. Verify sites for restriction digest and avoid double cuts using preferred cutter software 38. Heat-inactivate enzymes at appropriate temperature and duration following digestion according to manufacturer&#8217;s protocol. Minimal loss of DNA occurs during the linearization step.</li>
	<li>Methylate linearized plasmids using CpG methylase (M.Sssl) O/N at 30 &#176;C or leave untreated following manufacturer protocol except for the following adjustments.</li>
	<li>Perform 50 &#181;l reactions.</li>
	<li>Use 5 units of CpG methylase to methylate 700 ng of linearized plasmid.</li>
	<li>Run reactions O/N for 13-19 hr.</li>
	<li>Following CpG methylase reaction, perform DNA cleanup using standard, commercially available silica-gel membrane clean up kit according to manufacturer&#8217;s protocol. Significant losses of DNA occur following CpG methylase reaction, 30-60% loss.</li>
	<li>Verify methylation of plasmids via an Hpa II restriction digest.</li>
	<li>Take 1 &#181;g of methylated or non-methylated DNA and restriction digest with Hpa II for 1 hr&#160;at 37 &#176;C. Use 5-10 units of Hpa II for each &#181;g DNA. Following Hpa II digestion, perform DNA cleanup using preferred, commercially available silica-gel membrane clean up kit. Run both CpG methylated and non-methylated plasmids on a 1% agarose DNA gel in TAE or TBE buffer at 100 V for 45 min&#160;for visualization (Figure 4B).</li>
	<li>Subject both methylated and non-methylated plasmids to double digestion with appropriate restriction enzymes to release full-length luc 2 (vector) and CpG island (insert) fragments 38. Perform double digestion O/N at appropriate temperature and heat-inactivate enzymes following digestion according to manufacturer&#8217;s protocol. Minimal loss of DNA concentration occurs during double restriction digest.</li>
	<li>Run double digested plasmids on 1% DNA agarose gel at 100 V for 1 hr&#160;to allow for separation of vector and insert (Figure 4C). Caution. Wearing protective UV face shield, place DNA gel on a tabletop black light to visualize bands. Based on size, excise methylated and non-methylated insert and non-methylated vector using a clean surgical blade.</li>
	<li>Gel extract DNA using saturated phenol, pH 6.6. Briefly, weigh DNA gel containing vector or insert. Use 100 &#181;l of phenol per 0.1 gram of DNA gel. Homogenize DNA gel in phenol using glass dounce homogenizer.</li>
	<li>Add chloroform (using 1/5 of phenol volume) and shake samples for 20 sec. Incubate samples at RT for 2-3 min. Centrifuge for 15 min at max speed (16.1 x 1,000 x g) at 4 &#176;C.</li>
	<li>Remove aqueous solution and add 0.1X volume of 3 M sodium acetate and 2.5X volume of ethanol. Incubate samples for 1 hr at -80 &#176;C. Following incubation, remove ethanol and re-suspend DNA in 30 &#181;l of preferred buffer. Significant loss of DNA occurs following isolation of inserts and vectors, 30-50% loss.</li>
	<li>Re-ligate methylated and non-methylated inserts to non-methylated vector using T4 DNA ligase 38. Use a 1:4 ratio of vector to insert for ligation reactions. Setup reaction according to manufacturer instructions. Use a total volume of 50 &#181;l for reactions and incubate O/N at -20 &#176;C or on ice with lid over ice bucket.</li>
	<li>Following ligation, perform DNA cleanup using preferred, commercially available silica-gel membrane clean up kit. Significant loss of DNA occur following ligation reaction, an approximate 30-50% loss of DNA. Verify re-ligation by running on 1% agarose DNA gel at 100 V for 45 min&#160;and assess concentration of re-ligated plasmids (Figure 4D).</li>
	<li>Transfect methylated or non-methylated plasmids into D54 cells using a commercial transfection reagent according to manufacturer&#8217;s protocol. Seed D54 cells onto 12-well plate at a 0.14 x 106 cells/well.</li>
	<li>Following 24 hr, transfect cells with equal concentrations of either (1) non-methyated CpG-luc2 plasmid + Renilla or other control luciferase vector or (2) methylated CpG-luc2 plasmid + Renilla or other control luciferase vector.</li>
	<li>Allow cells to transfect for 24 hr&#160;before performing dual luciferase assay. Perform assay according to manufacturer instructions. Perform readings using a luminometer. Read each well in triplicate.</li>
	<li>Calculate ratio of Firefly luciferase activity to Renilla or other control luciferase activity. Normalize methylated Firefly:control luciferase activity to non-methylated Firefly:control luciferase activity by dividing methylated luciferase activity by non-methylated luciferase activity</li>
</ol>

The authors have no disclosures.

This protocol describes the isolation of an enriched population of astrocytes via FACS as well as a variety of techniques that allow for both correlative and causative studies between DNA methylation and gene expression. These techniques, used in isolation or in combination, are particularly useful for laboratories that work with tissue of high cellular heterogeneity or are interested in the DNA methylation status of a particular gene or gene region versus global DNA methylation changes. One relatively unique challenge i...

Epigenetics is the study of chemical modifications that can affect the transcriptional activity of the genome. Essentially, without a change in the DNA sequence, epigenetic modifications such as DNA methylation, histone acetylation, and histone methylation are sufficient to reversibly alter patterns of gene expression 1. DNA methylation, a potent regulator of gene expression, is the most well characterized epigenetic modification. DNA methylation is the covalent attachment of methyl groups on the C5 position of a cytosine, typically the cytosine of a cytosine-guanine dinucleotide, also known as a CpG site. Areas that contain a high density of CpG sites are known as CpG islands (CGIs). CGIs are frequently associated with transcriptional start sites (TSS) and gene promoters 1-3. Thus, while changes in DNA methylation at CGIs are not always concomitant with changes in cellular expression or function, changes in DNA methylation at CGIs can exert powerful regulation on transcriptional activity 2. 
Historically, DNA methylation was observed to be essential in embryogenesis, imprinting, and development, with little changes in the levels of DNA methylation occurring in post-mitotic cells (with the exception of alterations in cancer-related genes) 4,5. However, the field of neuroepigenetics has highlighted an important non-developmental role for DNA methylation. Specifically, cognitive epigenetics has redefined DNA methylation as a highly plastic mechanism integral in mediating both the transcriptional activation and repression of genes essential for the process of learning and memory 6. Apart from cognitive epigenetics, studies modeling ischemic injury and neuropathic pain characterize DNA methylation as a labile mechanism that responds rapidly to a variety of CNS insults 7-9. In regards to astrocytes, several lines of evidence suggest DNA methylation plays an important role in astrogliogenesis. Fan et al., found that conditional KO of DNMT1 in neural progenitor cells (NPCs) resulted in precocious development of astrocytes concordant with a global state of hypomethylation 10. Additionally, Perisic et al., concluded differential levels of DNA methylation of the GLT-1 promoter mediated differential levels of expression of the glutamate transporter in the cortex and cerebellum, emphasizing a role in DNA methylation in establishing brain-region specific patterns of astrocytic gene expression 11. Overall, numerous studies underscore the dynamic and labile nature of DNA methylation in the CNS as environment, drugs, and injury have all been shown to change DNA methylation and often, gene expression 4,9. Together, these neuroepigenetic studies point to DNA methylation as a feasible therapeutic target with the potential to mitigate a variety of CNS pathologies.
As the field of epigenetics expands its understanding of the role of DNA methylation in neurodevelopment and disease, the challenge of moving DNA methylation towards a therapeutic target is performing not only correlative, but causative studies that define specific gene targets and sites. Additionally, surveying changes in DNA methylation specific to brain region and cell type remains an ongoing and time worthy challenge unique to the field of neuroepigenetics. This protocol utilizes a variety of techniques including fluorescence-activated cell sorting (FACS) of astrocytes, methylation-sensitive high resolution melt analysis (MS-HRM), and a methylation luciferase assay to investigate the DNA methylation status of KCNJ10, a gene that encodes for Kir4.1. Kir4.1 is a glial specific potassium channel that demonstrates both brain region and cell specific patterns of expression in the CNS 12-16. Kir4.1 expression increases moving from rostral to caudal CNS regions, with the highest expression occurring in the spinal cord 15. Although the channel is expressed in ependymal cells, oligodendrocytes and their precursor cells, Kir4.1 is predominantly expressed in astrocytes and thought to be essential for maintaining homeostatic levels of potassium as well as supporting glutamate uptake by setting the astrocytic resting membrane potential at a hyperpolarized -80mV 12,16-19. Importantly, the expression of Kir4.1 is non-static both during development and following multiple forms of CNS injury 20-25. We wished to examine the epigenetic regulation of this channel, specifically in astrocytes during development. The techniques utilized offer gene-specific and targeted CpG site analyses that provide causal evidence for a role of DNA methylation in regulating KCNJ10 gene expression. These techniques can be applied to other genes.

<table><tbody><tr><td>Papain Dissociation System</td><td>Worthington Biochemical Corporation</td><td>LK003150</td><td></td></tr><tr><td>AllPrep DNA/RNA Mini Kit</td><td>Qiagen</td><td>80204</td><td></td></tr><tr><td>Methyl Primer</td><td>Applied Biosystems</td><td>online</td><td>software to localize CpG Islands</td></tr><tr><td>EZ DNA methylation Kit</td><td>Zymo Research</td><td>D5001</td><td></td></tr><tr><td>Rat Premixed Calibration Standard</td><td>EpiGenDx</td><td>80-8060R-Premix</td><td></td></tr><tr><td>CpG Methylase (M.Sssl)</td><td>Zymo Research</td><td>E2010</td><td></td></tr><tr><td>QIAquick Gel Extraction&amp;nbsp;</td><td>QIagen</td><td>28704</td><td>Used for gel extraction and DNA cleanup</td></tr><tr><td>Restriction enzymes</td><td>New England BioLabs</td><td></td><td></td></tr><tr><td>NEB cutter</td><td>New England BioLabs</td><td>online</td><td>verify restriction digest sites</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr><tr><td>Luc2 vector, pGL4.10</td><td>Promega</td><td>E6651</td><td></td></tr><tr><td>renilla vector, pGL4.74</td><td>Promega</td><td>E2241</td><td></td></tr><tr><td>TD-20/20 Luminometer</td><td>Turner Designs</td><td></td><td></td></tr><tr><td>Lipofectamine LTX and Plus Reagent</td><td>Life Technologies</td><td>A12621</td><td></td></tr><tr><td>Phenol, saturated pH 6.6/6.9</td><td>Fisher Scientific</td><td>BP 17501-100</td><td></td></tr><tr><td>Nanodrop 2000/2000c Spectrophtometer</td><td>ThermoScientific</td><td></td><td></td></tr><tr><td>MeltDoctor Master Mix</td><td>Life Technologies</td><td>4415440</td><td></td></tr><tr><td>High Resolution Melt (HRM) Software v2.0</td><td>Life Technologies</td><td>4397808</td><td></td></tr><tr><td>AB SDS software v2.3</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB High Resolution Melting Getting Started Guide&amp;nbsp;</td><td>Life Technologies</td><td>online</td><td></td></tr><tr><td>AB 7900HT Fast Real-Time System</td><td>Life Technologies</td><td></td><td></td></tr></tbody></table>

correlating gene-specific dna methylation changes with expression and transcriptional activity of astrocytic kcnj10 (kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

An enriched population of astrocytes was acquired via FACS sorting of eGFP-S100&#946; transgenic animals 27. Due to decreasing quality of cells and molecular molecules isolated from animals older than postnatal day 50 (p50), animals aged p0-p40 are optimal for such experiments. Cortical tissue was used for these experiments. Cortices from two to six animals were pooled together. FACS was performed at UAB Comprehensive Flow Cytometry Core facility. Sorting was performed on Becton Dickinson FacsAria II. eGFP exc...

Watch this Scientific Journal Video about Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1 at JoVE.com

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

correlating-gene-specific-dna-methylation-changes-with-expression

Research

JoVE Journal

Biology

7.9K Views.  University of Alabama at Birmingham. DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression. 

Video: Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
	In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

This study describes the procedures of setting up a novel neuronal axon and (astro)glia co-culture platform. In this co-culture system, manipulation of direct interaction between a single axon (and single glial cell) becomes feasible, allowing mechanistic analysis of the mutual neuron to glial signaling.

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Developmental Biology

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with enhanced [Ca2+]i signals. It has been proposed that astrocytes in turn regulate neurons and blood vessels through calcium-dependent mechanisms, such as the release of signaling molecules. However, [Ca2+]i imaging in entire astrocytes has only recently become feasible with genetically encoded calcium indicators (GECIs) such as the GCaMP series. The use of GECIs in astrocytes now provides opportunities to study astrocyte [Ca2+]i signals in detail within model microcircuits such as the striatum, which is the largest nucleus of the basal ganglia. In the present report, detailed surgical methods to express GECIs in astrocytes in vivo, and confocal imaging approaches to record [Ca2+]i signals in striatal astrocytes in situ, are described. We highlight precautions, necessary controls and tests to determine if GECI expression is selective for astrocytes and to evaluate signs of overt astrocyte reactivity. We also describe brain slice and imaging conditions in detail that permit reliable [Ca2+]i imaging in striatal astrocytes in situ. The use of these approaches revealed the entire territories of single striatal astrocytes and spontaneous [Ca2+]i signals within their somata, branches and branchlets. The further use and expansion of these approaches in the striatum will allow for the detailed study of astrocyte [Ca2+]i signals in the striatal microcircuitry.

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

The properties and functions of astrocyte intracellular Ca2+ signals in the striatum remain incompletely explored. We describe methods to express genetically encoded calcium indicators in striatal astrocytes using adeno-associated viruses of serotype 2/5 (AAV2/5), as well as procedures to reliably image Ca2+ signals within striatal astrocytes in situ.

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

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 ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

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