Ce travail a été financé par l&#39;Institut Max Planck de psychiatrie et de l&#39;Union européenne Marie Curie Réseau de formation initiale (contrat NINA 238 665).

<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>

Pas de conflits d&#39;intérêt déclarés.

Programmation épigénétique est de plus en plus reconnue comme un mécanisme important qui sous-tend la santé et la maladie. Nous présentons ici une méthode plus perfectionnée pour l&#39;isolement simultané de l&#39;ADN et d&#39;ARN à partir micropunches et les étapes ultérieures d&#39;obtenir des profils de méthylation d&#39;ADN par séquençage au bisulfite. Le flux de travail optimisé permet une analyse pratique de la programmation épigénétique dans le cerveau en réponse à des stimuli environnementaux. En outre, cette approche facilite l&#39;enquête de la relation fonctionnelle entre la méthylation de l&#39;ADN et l&#39;expression des gènes à la fois comme sont analysés à partir du même échantillon. Enfin, le nombre d&#39;animaux nécessaires pour l&#39;expérience peut être considérablement réduit. Certaines précautions et les directives doivent être suivies lors de l&#39;exécution du flux de travail présenté. Considérant l&#39;extraordinaire complexité et l&#39;hétérogénéité du cerveau et, depuis motif de méthylation et d&#39;expression peut varier d&#39;une manière cellules et des tissus spécifiques, il est de la plus hauteimportance que micropunching est effectuée de manière standardisée. Comme un cas au point, la programmation épigénétique de AVP est détecté dans le paravaventriuclar, mais pas dans le noyau supraoptique (figure 4b-d), deux Avp exprimer régions de l&#39;hypothalamus 4. À cet égard, la disponibilité de l&#39;ADN et l&#39;ARN du poinçon même tissu est avantageux que l&#39;expression d&#39;ARN de gènes marqueurs tissulaires certaines spécifiques peuvent être utilisés dans certains cas, pour certifier que la zone voulue a été frappé. Bien que micropunching peut réduire l&#39;hétérogénéité cellulaire, il ne faut pas oublier que cette approche ne conduise pas à l&#39;isolement de types de cellules simples ou des populations. Étapes de purification supplémentaires pourraient être envisagées pour traiter ce sujet. Pour le bisulfite de séquençage de conception d&#39;amorce optimale pour l&#39;amplification par PCR de bisulfite de traitement suivant est essentiel. Les produits de PCR de plus de 400 pb de longueur peut être problématique en raison de la dégradation de l&#39;ADN par le btraitement isulfite. Il convient également de noter que la PCR nichée peut donner des résultats biaisés si seulement quelques modèles intacts contribuer au processus d&#39;amplification. Paires d&#39;amorces qui sont spécifiques pour l&#39;amplicon modification doit être conçue; leurs séquences ne doivent pas contenir des dinucléotides CpG, afin d&#39;éviter la méthylation polarisée d&#39;amplification. En outre, des amorces doit contenir non-CpG cytosines pour éviter l&#39;amplification de l&#39;ADN non modifié ou incomplètement convertie. L&#39;amplification de certains modèles peuvent bénéficier de la conduite de démarrage à chaud PCR. Dans tous les cas, la pertinence de paires d&#39;amorces doivent être vérifiées dans des expériences pilotes de manière à empêcher la détérioration de la valeur des spécimens expérimentaux. Séquençage bisulfite représente une méthode standard pour l&#39;analyse de méthylation spécifique de site tel qu&#39;il est capable de détecter de manière fiable et précise de la méthylation de CpG uniques résidus de manière spécifique de l&#39;allèle 8. Le séquençage direct du produit de PCR n&#39;est pas conseillé que la méthylation mixte oFA CpG résidus apparaît comme un pic C / double T dans le électrophérogramme, ce qui peut empêcher la quantification de la méthylation du site concerné. Pour cette raison, une étape de clonage intermédiaire est effectuée et plusieurs clones sont séquencés (~ 20-30) pour déterminer l&#39;étendue de la méthylation. Pyroséquençage représente une méthode alternative pour quantifier méthylation de l&#39;ADN. Il utilise également la conversion au bisulfite et le bisulfite de PCR mais au lieu de clonage et le séquençage de l&#39;amplicon est soumis à une réaction directe pyroséquençage où le statut de méthylation d&#39;un résidu CpG est lu comme un C / T polymorphisme de nucléotide unique qui peut être facilement quantifiés. Les deux méthodes de détecter des niveaux similaires de méthylation 9, mais depuis le clonage étape peut être omise pyroséquençage prend plus de temps efficace. Toutefois, selon le modèle de seulement 40 à 100 nucléotides peuvent être séquencés à la fois de sorte que plusieurs séries de pyroséquençage avec amorce de séquençage différentes sont nécessaires. Il s&#39;agit d&#39;une limitation critique, étant donné thamontants plus élevés de l&#39;ADN t (environ 1 pg par réaction) sont nécessaires, ce qui prévisible dépasse les montants disponibles à partir de minuscules coups de tissus cérébraux. Ainsi la première analyse des régions dans la gamme de plusieurs kilobases par pyroséquençage semble moins adapté que la lecture seul clone. Néanmoins, à la suite de l&#39;identification d&#39;une petite région de pyroséquençage intérêt doit être pris en considération. Collectivement, le protocole décrit ci-dessus fournit une approche précise, efficace et rationalisé pour l&#39;analyse rapide et rentable de micropunches cerveau de modifications épigénétiques. Spécimens multiples peuvent être traitées en un seul passage et les méthodes sont adaptées lors de l&#39;exécution des échantillons provenant de taille intermédiaire expériences.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Sang DNeasy &amp; Kit de tissus </td><td> Qiagen </td><td> 69506 </td></tr><tr><td> Nucleospin II de l&#39;ARN </td><td> Macherey-Nagel </td><td> 740955,50 </td></tr><tr><td> EpiTect Kit bisulfite </td><td> Qiagen </td><td> 59104 </td></tr><tr><td> Nucleospin Extrait </td><td> Macherey-Nagel </td><td> 740609,50 </td></tr><tr><td> pGEM-T Vector kit de clonage </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 séquençage Clean-Up </td><td> Millipore </td><td> LSK09624 </td></tr><tr><td> Hotstar polymérase Taq </td><td> Qiagen </td><td> 203203 </td></tr><tr><td> Taq Polymérase </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

L&#39;exposition à l&#39;alimentation, les médicaments et l&#39;adversité au cours de la vie au début fenêtres sensibles de la vie 1,2 peut conduire à des changements durables dans l&#39;expression des gènes qui contribuent à l&#39;affichage des phénotypes physiologiques et comportementaux. Cette programmation de l&#39;environnement est susceptible d&#39;augmenter la susceptibilité à métaboliques, les maladies cardio-vasculaires et mentales 3,4. Méthylation de l&#39;ADN et des modifications des histones sont considérés comme des processus clés dans la médiation du dialogue gènes-environnement et semblent aussi sous-tendaient la programmation de l&#39;environnement 5. Chez les mammifères, méthylation de l&#39;ADN comprend typiquement l&#39;addition covalente par un groupe méthyle à la position 5 de la cytosine dans le contexte de dinucléotides CpG. Méthylation de CpG se produit de façon hautement tissu-spécifique de cellule et qui en fait un défi à étudier, des petites régions discrètes du cerveau, où l&#39;hétérogénéité cellulaire est haute et la quantité de tissu limitée. En outre, barce que l&#39;expression des gènes et de méthylation sont étroitement liés les événements, une valeur accrue peut être obtenue en comparant les deux paramètres dans le même échantillon. Ici, un protocole étape par étape (Figure 1) pour l&#39;enquête de la programmation épigénétique dans le cerveau est présenté en utilisant le paradigme du «séparation maternelle» de l&#39;adversité début de la vie à des fins d&#39;illustration. Le protocole décrit la préparation de micropunches de cerveaux de souris différentiellement d&#39;âge à partir duquel l&#39;ADN et l&#39;ARN peuvent être simultanément isolés, permettant ainsi méthylation de l&#39;ADN et les analyses d&#39;expression génique dans le même échantillon.

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

Un flux de travail simplifié pour étudier méthylation de l&#39;ADN et des modifications d&#39;expression des gènes lors de début de la vie de stress est montré. A partir de la séparation maternelle des souriceaux nouveau-nés et l&#39;isolement des tissus cérébraux distincts, nous représentons un protocole à la fois isoler l&#39;ADN et l&#39;ARN à partir de poinçons de tissus cérébraux pour le séquençage du bisulfite de suite et analyse RT-PCR.

Analyse de la méthylation de l&#39;ADN Optimisé et d&#39;expression de gènes à partir de petites zones définies anatomique du cerveau

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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Neuroscience

18.7K vues.  Max Planck Institute of Psychiatry. Un flux de travail simplifié pour étudier méthylation de l'ADN et des modifications d'expression des gènes lors de début de la vie de stress est montré. A partir de la séparation maternelle des souriceaux nouveau-nés et l'isolement des tissus cérébraux distincts, nous représentons un protocole à la fois isoler l'ADN et l'ARN à partir de poinçons de tissus cérébraux pour le séquençage du bisulfite de suite et analyse RT-PCR.

Vidéo: 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

La moelle épinière électrophysiologie II: fabrication des électrodes extracellulaires d&#39;aspiration

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

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Neurosciences

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.

Isoler les cellules souches nasal olfactif des rongeurs ou des êtres humains

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.

Imagerie à haute résolution en direct du comportement des cellules dans le développement neuro-épithélium

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.

Chirurgie stéréotaxique pour lésion excitotoxique des zones spécifiques du cerveau chez le rat adulte

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.

Microdissection par capture laser de populations enrichies de neurones neurones isolés ou pour l&#39;analyse d&#39;expression génique après un traumatisme crânien

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.

Micromanipulation de l&#39;expression génique dans le cerveau de poisson zèbre adulte en utilisant microinjection Cerebroventricular d&#39;oligonucléotides Morpholino

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

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

ARN-Seq analyse de l&#39;expression génique différentielle dans électroporation embryon de poulet de la moelle épinière

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

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.

Analyse des modifications d&#39;expression génique dans l&#39;hippocampe de rat après stimulation cérébrale profonde de l&#39;antérieur thalamique Nucleus

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.

Préparation de tranches de cerveau aiguë en utilisant un optimisé N-méthyl-D-glucamine méthode de recouvrement protecteur

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